JP6953435B2 - Dimer compound - Google Patents

Description

The present invention relates to dimeric compounds that act as inhibitors of SMAC proteins that bind to apoptosis inhibitory proteins (IAPs) and / or inhibitors of activated caspase proteins that bind to IAPs. These molecules are useful for ameliorating, treating or controlling cancer, especially solid tumors. They can be combined with other agents such as DR5 / FAP agonist antibodies.

These compounds bind to the BIR2 or BIR2 and BIR3 regions of the IAP protein, including XIAP and cIAP, resulting in activation or reactivation of the caspase cascade and are therefore useful in the treatment of proliferative disorders, including cancer. ..

Cancer is a disease of uncontrolled cell proliferation that causes local growth of tumors and can cause distant metastases. One of the growth mechanisms of cancer cells is the avoidance of apoptosis, that is, programmed cell death. Alterations in the apoptotic pathway are associated with the resistance of cancer cells to standard therapies, such as chemotherapy or radiation therapy, as well as the development and progression of cancer. See, for example, E. Dean et al., "X-linked inhibitor of apoptosis protein as a therapeutic target," Expert Opin. Ther. Targets (2007) 11 (11): 1459-1471.

The two basic pathways for apoptotic cell death are the endogenous and extrinsic pathways. The endogenous apoptotic pathway can be initiated by a variety of mechanisms, including cell stress and drug-induced DNA damage. The extrinsic pathway can be initiated by activation of the death receptor by chemokines. Initiation of either pathway results in activation of a family of proteases called caspases. Once activated, the caspase can act to cleave various substrates that produce a cascade of events that result in activation of effector caspases 3 and 7, as well as eventual cell death. The IAP family of proteins can bind to caspases and inhibit their activity, thus inhibiting apoptosis. See, for example, Dean, 1460 above.

The IAP can include up to three copies of the baculovirus IAP repeat (BIR) domain, BIR1, BIR2, and homologous structure domains called BIR3. The BIR3 domains of the prototype IAPs cIAP and XIAP can bind to and inhibit activated caspase-9. On the other hand, the BIR2 domain binds to and inhibits caspases 3 and 7. The pro-apoptotic protein Smac (also known as DIABLO) can block the BIR2 and BIR3 domains of IAPs that compete with activated caspases, resulting in the release of activated caspases from the IAP and an apoptotic program. To complete. See, for example, S. Wang, "Design of Small-Molecule Smac Mimetics as IAP Antagonists," Current Topics in Microbiology and Immunology 348, DOI 10.1007 / 82_2010_111, pp. 89-113.

Peptides and small molecules have been reported to bind to the BIR3 region of XIAP and cIAP to mimic the action of Smac proteins and release activated caspases. See, for example, Dean and M. Gyrd-Hanse et al., "IAPs: From caspase inhibitors to modulators of NF-κB, inflammation and cancer," Nature Review / Cancer, August 2010, Vol 10: 561-574. ..

XIAP inhibitors can strongly sensitize pre-resistant human cancer cell lines to DR5-induced apoptosis.

WO 2014060770 relates to novel bicyclic heterocycles that may be antagonists of the IAP family of proteins (IAP). WO 2014161845 describes bispecific antibodies comprising at least one DR5-specific antigen binding site and at least one FAP-specific antigen binding site, DR5-specific antibodies, and their production. With respect to methods for, pharmaceutical compositions containing said antibodies, and their use.

In the art, finding effective treatments for targeted cancers, using XIAP inhibitors alone or in combination with DR5 / FAP agonist antibodies, remains a problem.

又はその薬学的に許容される塩であり、ここで、Ｌ、Ａ及びＲ_１は本明細書に記載されるものである。 One aspect of the present invention is Formula I:

Or a pharmaceutically acceptable salt thereof, wherein L, A and R ₁ are those described herein.

The present invention also relates to a pharmaceutical composition comprising one or more compounds of the invention or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers or additives.

The invention further relates to methods of ameliorating, controlling or treating cancers, including solid tumors, such as lung, pancreas, colon, breast, bone, and prostate cancer, especially in mammals, especially humans. It comprises administering to the mammal a therapeutically effective amount of the pharmaceutically acceptable salt.

N-(2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S)) in the cell viability assay of SK-OV-3 cells -2- (Methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl)- 3-Methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indoline-3-carboxamide trihydro Shows single-drug activity of chloride. N- (2-(((S)-)-in a cell viability assay of HCT-116 cells in combination with a single agent and the DR5-FAP bispecific antibody XAB (gradual increase in FAP-DR5 concentration). 3-((2,6-difluorophenyl) carboxamide) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) ) Acetyl) Isoindoline-5-yl) Amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-methyl-2- ( It shows the activity of ((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indoline-3-carboxamide trihydrochloride. N- as a combination of a single agent and the DR5-FAP bispecific antibody XAB (fixed concentration of FAP-DR5, 1.5 μg / ml) in the cell viability assay of HCT-116 cells. (2-(((S) -3-((2,6-difluorophenyl) carboxamide) -2-((S) -2-((S) -2- (methylamino) propanamide) -2-( Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) )-5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) Indolin-3-carboxamide Shows the activity of trihydrochloride. A schematic diagram of the FAP-DR5 bispecific antibody molecular design and mechanism of action is shown.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as would be commonly understood by one of ordinary skill in the art to which the present invention belongs.

The following terms used herein have the following definitions:

The term "C _1-6 -alkyl" means a monovalent linear or branched saturated hydrocarbon with 1 to 6 carbon atoms, alone or in combination with other groups. Examples include methyl (Me), ethyl (Et), propyl, isopropyl, butyl (also known as n-butyl), iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

The term "C _1-6 -alkoxy" may be linear or branched, or may have single or multiple branches, alone or in combination with other groups-OC. _{Representing a 1-6} -alkyl group, where the alkyl group generally contains 1 to 6 carbon atoms, for example, this is methoxy (OME, MeO), ethoxy (OEt), propoxy, isopropoxy ( i-propoxy), n-butoxy, i-butoxy (iso-butoxy), 2-butoxy (sec-butoxy), t-butoxy (tert-butoxy), isopentyloxy (i-pentyloxy) and the like can be mentioned. A particular "C _1-6 -alkoxy" is a group having 1 to 4 carbon atoms, especially 2-methoxypropanol.

The term "C _1-6 -alkoxy-C _{1-6 -alkyl" is substituted by "C 1-6} -alkoxy" as described herein, alone or in combination with other groups. Means "C _1-6 -alkyl" as described herein. Examples include -C (H, Me) -OMe and the like.

The term "halogen" means an atom selected from F, Cl, Br or I. In certain embodiments, halogen means F.

The term "C _3-6 -heterocycloalkyl" refers to 3 to 6 members having at least one O, N or S, especially one O heteroatom, alone or in combination with other groups. Refers to the carbon ring. A specific example is tetrahydropyranyl.

The term "half inhibitory concentration" _{(IC 50)} means the concentration of a particular compound required to obtain 50% inhibition of the biological process in vitro. The IC ₅₀ value can be logarithmically converted to the pIC ₅₀ value (-log IC ₅₀ ), where the higher the value, the greater the exponential effect. The IC ₅₀ value, not absolute value, experimental conditions, depending on the concentration used, for example. The IC ₅₀ value can be converted to an absolute inhibition constant (Ki) using the Cheng-Prusoff equation (Biochem Pharmacol (1973) 22: .. 3099). Pharmacol. 1973: 22:30 99. The term "inhibition constant (Ki)" indicates the absolute binding affinity of a particular inhibitor for a receptor. It is measured using a competitive binding assay and is equal to the concentration at which a particular inhibitor occupies 50% of the receptor in the absence of a competitive ligand (eg, radioligand). The Ki value can be logarithmically converted to a pKi value (-log Ki), and the higher the value, the greater the exponential effect.

"Therapeutically effective amount" means the amount of compound sufficient to provide such treatment for a medical condition when administered to a subject to treat the medical condition. The "therapeutically effective amount" is the compound, the medical condition to be treated, the severity or disease to be treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other matters. It will change depending on the factors.

"Pharmaceutically acceptable" of a pharmaceutically acceptable carrier, additive, etc. means that a particular compound is pharmacologically acceptable and substantially non-toxic to the subject to whom it is administered. means.

A "pharmaceutically acceptable salt" is formed from a suitable non-toxic organic or inorganic acid or organic or inorganic base that retains the biological efficacy and properties of the compounds of the invention. Refers to a general acid addition salt or base addition salt. Examples of acid addition salts are those derived from inorganic acids such as hydrochloride, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitrate, as well as organic acids such as p-toluenesulfone. Includes those derived from acids, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, trifluoroacetic acid and the like. Examples of base addition salts include those derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides, such as tetramethylammonium hydroxide. Chemical modification of a pharmaceutical compound (ie, a drug) to a salt is a well-known technique for pharmaceutical chemists to obtain improvements in the physical and chemical stability, hygroscopicity, fluidity and solubility of the compound. Is. See, for example, Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (1995), pp. 456-457.

As used in the present application, if the formula or group lacks a substituent, it is presumed that the valence is not completely satisfied and the missing substituent is H.

Whenever an asymmetric carbon is present in a chemical structure, all stereoisomers associated with that asymmetric carbon are intended to be included by structure as pure stereoisomers and mixtures thereof.

The present invention also provides a pharmaceutical composition, a method of using the above compound and a method of preparing the above compound.

The term "bispecific antibody that specifically binds to Death Receptor 5 (DR5) and fibroblast-activating protein (FAP)" is a diagnostic agent in targeting cells expressing DR5 and FAP. / Or refers to a bispecific antibody capable of binding DR5 and FAP with sufficient affinity to be useful as a therapeutic agent. In particular, "bispecific antibodies that specifically bind to Death Receptor 5 (DR5) and Fibroblast Activating Protein (FAP)" target DR5 on tumor cells and FAP in the substrate surrounding the tumor. Refers to bispecific antibodies. In one embodiment, the degree of binding of a bispecific antibody that specifically binds a desreceptor 5 (DR5) and a fibroblast activation protein (FAP) to an unrelated, non-FAP or non-DR5 protein is, for example, enzymatic binding. Less than about 10% of antibody binding to DR5 or FAP as measured by immunosorbent assay (ELISA), surface plasmon resonance (SPR) based assay (eg Biacore) or flow cytometry (FACS). In certain embodiments, bispecific antibodies that specifically bind to Death Receptor (DR5) and Fibroblast Activating Protein (FAP) are ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM. , ≦ 0.01 nM, or ≦ 0.001 nM (e.g. ^{10 -8} M or less, for example, ^{10 -8} M from ^{10 -13} M, such as ¹⁰ from ^-9 M ^{10 -13} M) having a dissociation constant of (Kd). In certain embodiments, bispecific antibodies that specifically bind to Death Receptor 5 (DR5) and Fibroblast Activating Protein (FAP) are conserved between DR5 or FAP from different species DR5 or FAP. Binds to the epitope of. Preferably, the bispecific antibody binds to human and cynomolgus monkey DR5 and human, cynomolgus monkey and mouse FAP.

The term "antibody that specifically binds to the death receptor (DR5)" refers to DR5 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in the targeting of cells expressing DR5. Refers to an antibody that can bind. In one embodiment, the degree of binding of the death receptor (DR5) to an irrelevant, non-DR5 protein is, for example, the binding of the antibody to DR5 when measured by radioimmunoassay (RIA) or flow cytometry (FACS). It is less than about 10% of. In certain embodiments, the antibody that binds DR5 is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (eg, ^10-8 M or less, eg, 10). ^{It has} a dissociation constant (Kd) of -8 M to ^10-13 M, eg ^10-9 M to ^{10-13 M).} In certain embodiments, an antibody that specifically binds to death receptor 5 (DR5) binds to an epitope of DR5 that is conserved between DR5s from different species. In particular, the antibody binds to human and cynomolgus monkey DR5. The term "antibody that specifically binds to Death Receptor 5 (DR5)" also includes bispecific antibodies capable of binding DR5 to a second antigen.

The term "antibody" as used herein is used in the broadest sense and includes, but is not limited to, various antibody structures, including monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies). ) And an antibody fragment as long as it exhibits the desired antigen-binding activity.

The term "antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F (ab') ₂ ; diabody, cross-Fab fragment; linear antibody; single chain antibody molecule (eg scFv). And include multispecific antibodies formed from antibody fragments. The scFv antibody is described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, antibody fragments can assemble at functional antigen binding sites with VH domain properties, i.e., with VL domains, or with VL domain properties, i.e. with VH domains. Includes a single-chain polypeptide that provides the antigen-binding properties of a full-length antibody.

The term "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL) and a VH domain and a first constant domain of a heavy chain (CH1). One embodiment of a bispecific antibody of the invention comprises at least one Fab fragment, where either the variable or constant regions of the heavy and light chains are exchanged. By exchanging either the variable region or the constant region, the Fab fragment is also referred to as a "crossed Fab fragment" or "xFab fragment" or a "crossover Fab fragment". Two different chain compositions of crossover Fab molecules are possible and are included in the bispecific antibodies of the invention. In contrast, the variable regions of the Fab heavy chain and light chain have been exchanged, that is, the crossover Fab molecule has a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), and a heavy chain variable. It contains a peptide chain consisting of a region (VH) and a light chain constant region (CL). This crossover Fab molecule is also called _{CrossFab (VLVH).} On the other hand, when the constant regions of Fab heavy chain and light chain are exchanged, the crossover Fab molecule is composed of a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL), and a light chain variable region. It contains a peptide chain consisting of (VL) and a heavy chain constant region (CH1). This crossover molecule is also called _{CrossFab (CLCH1).} Bispecific antibody formats containing crossover Fab fragments are, for example, International Publication Nos. 2009/08052, 2009/08253, 2009/08251, 2009/08254, 2010/136172. , 2010/145792 and 2013/026831.

The term "single chain Fab fragment" or "scFab" refers to antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL) and A polypeptide consisting of a linker, wherein the antibody domain and the domain are described below in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH- It has one of CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; where the linker is from at least 30 amino acids, preferably from 32. It is a polypeptide of amino acids between 50. The single chain Fab fragment a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 and d) VL-CH1-linker- VH-CL is stabilized via a natural disulfide bond between the CL domain and the CH1 domain. In addition, these single chain Fab molecules are further stabilized by the formation of interchain disulfide bonds via the insertion of cysteine residues (eg, the 44th position of the variable heavy chain and the 100th position of the variable light chain by Kabat numbering). May be transformed.

The term "N-terminal" means the last amino acid at the N-terminus. The term "C-terminal" means the last amino acid at the C-terminal.

The phrase "antibody that binds to the same epitope" as a reference antibody refers to an antibody that inhibits the binding of a reference antibody to its antigen by 50% or more in a competitive assay, and conversely, a reference antibody is that antigen of an antibody in a competitive assay. Inhibits binding to 50% or more. An exemplary competitive assay is provided herein.

The term "antigen-binding domain" refers to a portion of an antibody-binding molecule that specifically binds to part or all of an antigen and comprises a region that is complementary to part or all of the antigen. When the antigen is large, the antigen-binding molecule can only bind to a specific portion of the antigen called an epitope. The antigen binding domain may be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term "chimeric" antibody is one in which a portion of the heavy chain and / or light chain is derived from a particular origin or species, while the rest of the heavy chain and / or light chain is of a different origin or usually prepared by recombinant DNA technology. Refers to an antibody derived from a species. Chimeric antibodies comprising a rabbit variable region and a human constant region are preferred. Other preferred forms of the "chimeric antibody" encapsulated by the present invention are those in which the constant region has been modified or the original antibody to give rise to the properties according to the invention, especially with respect to C1q binding and / or Fc receptor (FcR) binding. It has been changed from. Such chimeric antibodies are also referred to as "class switch antibodies." A chimeric antibody is a product of an expressed immunoglobulin gene that comprises a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region. Methods for making chimeric antibodies include common recombinant DNA, and gene transfection techniques are well known in the art. See, for example, Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.

The term "effector function" refers to the biological activity that can result from the Fc region of an antibody, which depends on the isotype of the antibody. Examples of antibody effector function are: C1q binding and complement-dependent cell injury (CDC); Fc receptor binding; antibody-dependent cell-mediated cell injury (ADCC); antibody-dependent cell phagocytosis (ADCP); cytokine secretion; Includes immune complex-mediated antigen uptake by antigen-presenting cells; downregulation of cell surface receptors (eg, B cell receptors); and activation of B cells.

The term "modified, modified, modified" is considered to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Modifications include modification of amino acid sequences, glycosylation patterns, or side chain groups of individual amino acids, and a combination of such techniques.

As used herein, the term "amino acid mutation" is intended to include substitutions, deletions, insertions, and modifications of amino acids. Any combination of substitutions, deletions, insertions, and modifications assumes that the final construct retains the desired properties, eg, reduced binding to the Fc receptor, or increased association with another peptide. It can be done to reach the final construct. Deletions and insertions of amino acid sequences include deletions of amino and / or carboxy terminus, as well as insertion of amino acids. A particular amino acid mutation is an amino acid substitution. Non-conservative amino acid substitutions, i.e., substitution of one amino acid with another amino acid having different structural and / or chemical properties, is particularly preferred for the purpose of altering the binding properties of the Fc region and the like. Amino acid substitutions include replacement of 20 standard amino acids (eg, 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) with naturally occurring amino acid derivatives or non-naturally occurring amino acids. .. Amino acid variants can be produced using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenicity, PCR, gene synthesis and the like. Methods other than genetic modification, such as modification of the side chain groups of amino acids by chemical modification, may also be useful. Various notations are used herein to indicate the same amino acid mutation. For example, substitution of proline at position 329 of the Fc domain to glycine is shown _{329G, G329, G 329, P329G} , or as Pro329Gly.

The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes the native sequence Fc region and the mutant Fc region. Although the boundaries of the Fc region of an IgG heavy chain can vary slightly, the human IgG heavy chain Fc region is usually defined as extending from Cys226 or Pro230 to the carboxyl end of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc or constant region is Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda. , MD, 1991, according to the EU numbering system, also known as the EU index. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the "subunit" of the Fc domain comprises one of the two polypeptides forming the dimeric Fc domain, namely the C-terminal constant region of the immunoglobulin heavy chain, and has a stable self-association ability. Refers to a polypeptide having. For example, subunits of the IgG Fc domain include IgG CH2 and IgG CH3 constant domains.

The phrase "modification that promotes association of the first and second subunits of the Fc domain" reduces or suppresses the formation of homodimers by association of a polypeptide containing the subunits of the Fc domain with the same polypeptide. It means manipulation of the peptide backbone or post-translational modification of the Fc domain subunit. The association-promoting modifications used herein are, in particular, separate for each of the two Fc domain subunits that are desired to be associated (ie, the first and second subunits of the Fc domain). These modifications are complementary to each other to facilitate association of the two Fc domain subunits. For example, association-promoting modifications can make those associations sterically or electrostatically preferred, respectively, by altering the structure or charge of one or both of the Fc domain subunits. Thus, (hetero) dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, and these polypeptides are sub. Additional components fused to each of the units (eg, antigen binding moieties) can be non-identical in the sense that they are not the same. In some embodiments, association-promoting modifications include amino acid mutations within the Fc domain, particularly amino acid substitutions. In certain embodiments, association-promoting modifications involve separate amino acid mutations, especially amino acid substitutions, in each of the two subunits of the Fc domain.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and have a structure that is substantially similar to that of a native antibody, or herein. Refers to an antibody having a heavy chain containing an Fc region defined in.

The term "human antibody" refers to an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human origin utilizing a repertoire of human antibodies or a sequence encoding another human antibody. To have. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues. As also referred to with respect to chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein is particularly referred to as, for example, "class switch", i.e., a change or mutation in the Fc moiety (eg, from IgG1). Also included are such antibodies that are modified with constant regions for C1q binding and / or FcR binding by IgG4 and / or IgG1 / IgG4 mutations) to produce the properties according to the invention.

The term "humanized" antibody refers to a chimeric antibody that comprises a non-human HVR amino acid residue and a human FR amino acid residue. In certain embodiments, the humanized antibody comprises substantially all of at least one, typically two variable domains, and all or substantially all of the HVR (eg, CDR) of the non-human antibody. Corresponding to, all or substantially all of FR corresponds to those of human antibodies. The humanized antibody may optionally include at least a portion of the antibody constant region derived from the human antibody. The "humanized" form of an antibody, eg, a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which constant regions are additionally modified to give rise to the properties according to the invention, especially with respect to C1q binding and / or Fc receptor (FcR) binding. It is a modification of the original antibody.

^１ 表ＡのすべてのＣＤＲの定義の番号付けは、Ｋａｂａｔら（以下参照）によって規定された番号付けの慣習に従っている。
^２ 表Ａにおいて使用されている小文字の「ｂ」を含む「ＡｂＭ」は、Ｏｘｆｏｒｄ Ｍｏｌｅｃｕｌａｒの「ＡｂＭ」抗体モデル化ソフトウェアによって定義されるＣＤＲを指す。 As used herein, the term "hypervariable region" or "HVR" is an antibody variable domain in which the sequence is hypervariable and / or forms a structurally defined loop ("hypervariable loop"). Refers to each area of. In general, native 4-chain antibodies include three HVs (H1, H2, H3) and three VLs (L1, L2, L3), for a total of six HVRs. HVRs generally contain amino acid residues from hypervariable loops and / or complementarity determining regions (CDRs), the latter having the highest sequence variability and / or being involved in antigen recognition. .. Exemplary hypervariable loops are amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101. (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are 24-34 for amino acid residues L1, 50-56 for L2, 89 for L3. -97, H1 31-35B, H2 50-65, and H3 95-102 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Hypervariable regions (HVRs) are also referred to as complementarity determining regions (CDRs), and these terms are used interchangeably herein with reference to the portion of the variable region that forms the antigen-binding region. Such specific areas are described in Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J Mol Biol 196: 901-917 (1987). ), The definition of which includes duplicates or subsets of amino acid residues when compared to each other. Nevertheless, where any definition applies with reference to the CDRs of an antibody or variant thereof, it is intended to be within the scope of the terms defined and used herein. Appropriate amino acid residues, including the CDRs defined by each of the above references, are specified in Table A below for comparison. The exact number of residues that contain a particular CDR will vary depending on the sequence and size of the CDR. One of ordinary skill in the art can routinely determine which residue contains a particular CDR, given the amino acid sequence of the variable region of the antibody.

^1. The numbering of all CDR definitions in Table A follows the numbering conventions specified by Kabat et al. (See below).
² "AbM", including the lowercase "b" used in Table A, refers to the CDR defined by Oxford Molecular's "AbM" antibody modeling software.

Kabat et al. Also defined a variable region sequence numbering system applicable to any antibody. One of ordinary skill in the art can explicitly assign this "Kabat numbering" system to any variable region sequence without relying on experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system defined by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise stated, references to the numbering of the positions of specific amino acid residues within the antibody variable region follow the Kabat numbering system.

With the exception of CDR1 of VH, CDRs generally contain amino acid residues that form a hypervariable loop. CDRs include "specificity-determining residues" or "SDRs" that are residues that come into contact with the antigen. The SDR is contained within a region of the CDR, abbreviated (abbreviated-) CDR, or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are amino acid residues L1. 31-34, L2 50-55, L3 89-96, H1 31-35B, H2 50-58, and H3 95-102. (See Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008).) Unless otherwise stated, HVR residues and other residues within the variable domain (eg, FR residues) are referred to herein. Numbered according to Kabat et al. Above.

An "isolated" antibody is one that has been isolated from its natural environment components. In some embodiments, the antibody is determined, for example, by electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse phase HPLC). If so, it is purified to a purity greater than 95% or 99%. See, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007) for a review of antibody purity assessment methods.

As used herein, the term "monoclonal antibody" means an antibody obtained from a substantially homogeneous population of antibodies, i.e., for example, containing naturally occurring mutations or during the production of monoclonal antibody preparations. The individual antibodies that make up the population are identical and / or bind to the same epitope, except for variant antibodies that occur and may generally be present in small amounts. In contrast to polyclonal antibody preparations, which usually contain different antibodies against different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is for a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention include, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and transgenic animals containing all or part of the human immunoglobulin loci. These methods and other exemplary methods for making monoclonal antibodies can be made by a variety of techniques, including methods utilized, which are described herein.

The term "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or radiolabel. The naked antibody may be present in the pharmaceutical formulation.

The term "natural antibody" refers to naturally occurring immunoglobulin molecules of various structures. For example, a native IgG antibody is a heterotetraglycoprotein of approximately 150,000 daltons consisting of two identical light chains disulfide-bonded and two identical heavy chains. Each heavy chain has a variable region (VH), also called a variable heavy chain domain or a heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain from the N-terminus to the C-terminus. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The term "package insert" is used to refer to the instructions typically included in the product packaging of a therapeutic product, and contraindications and / or contraindications to the indications, dosage, dosage, administration, combination therapy, use of such therapeutic products. Contains information about notes.

The phrase "substantially non-cross-reactive" refers to an antigen in which the molecule (eg, an antibody) differs from the actual target antigen of the molecule, especially when compared to the target antigen (eg, an antigen that is closely related to the target antigen). ) Is not recognized, or it means that it specifically binds to it. For example, an antibody can bind less than about 10% to less than about 5% to an antigen different from the actual target antigen, or about 10%, 9%, 8% to an antigen different from the actual target antigen. An amount consisting of 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or less than 0.1%, preferably about 2%, 1% or less. It can bind to less than 0.5%, most preferably less than about 0.2% or less than 0.1% of the actual target antigen, which is different from the actual target antigen.

The phrase "percent (%) amino acid sequence identity" for a reference polypeptide sequence is sequence identity with any conservative substitution after aligning the sequences and introducing gaps if necessary to obtain maximum percent sequence identity. Defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues of the reference polypeptide, if not considered part of. Alignments for the purpose of determining percent amino acid sequence identity are publicly available by various methods within the skill of the art, such as BLAST, BLAST-2, ALIGN, or Megaligin (DNASTAR) software. It can be achieved using possible computer software. One of ordinary skill in the art can determine the appropriate parameters for aligning the sequences, including any algorithm required to achieve maximum alignment over the overall length of the sequences being compared. However, for the purposes here,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech and source code is from the US Copyright Office, Washington, D.C. C. , 20559, along with user documentation, and is registered under US Copyright Registration Number TXU51087. ALIGN-2 is publicly available from Genentech (South San Francisco, California) or may be compiled from its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including V4.0D of digital UNIX. All sequence comparison parameters are set by the ALIGN-2 program and do not fluctuate.

In situations where ALIGN-2 is used for amino acid sequence comparison,% amino acid sequence identity of a given amino acid sequence A with or with respect to a given amino acid sequence B (or with respect to a given amino acid sequence B). A given amino acid sequence A that has or contains some percentage of amino acid sequence identity) is calculated as follows:
100 x fraction X / Y,
Here, X is the number of amino acid residues of the scores matched by the sequence alignment program ALIGN-2 as identical in the alignment of that program of A and B, and Y is the total number of amino acid residues of B. It will be appreciated that if the length of amino acid sequence A differs from the length of amino acid sequence B, then the% amino acid sequence identity of A to B is different from the% amino acid sequence identity of B to A. Unless otherwise stated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

Unless otherwise stated, the term "death receptor (DR5)" as used herein is derived from any vertebrate, including mammals such as primates (eg humans) and rodents (eg mice, rats). Refers to any natural DR5 of. The term includes "full length", untreated DR5 as well as any form of DR5 resulting from intracellular processing. The term also includes naturally occurring variants of DR5, such as splice variants or allelic variants. An exemplary human DR5 amino acid sequence is described in WO 2011/039126.

Unless otherwise stated, the term "fibroblast-activating protein (FAP)" as used herein is optional, including mammals such as primates (eg, humans) and rodents (eg, mice, rats). Refers to any native FAP of vertebrate origin. The term includes "full-length", untreated FAPs as well as any form of FAP resulting from intracellular processing. The term also includes naturally occurring variants of FAP, such as splice variants or allelic variants. Preferably, the anti-FAP antibody of the invention binds to the extracellular domain of FAP. The amino acid sequence of an exemplary FAP polypeptide sequence, including the sequence of human FAP, is disclosed in WO 2012/020006.

As used herein, cancer is a proliferative disorder such as cancer, such as colonic rectal cancer, sarcoma, head and neck cancer, squamous cell carcinoma, breast cancer, pancreatic cancer, gastric cancer. , Non-squamous lung cancer, small cell lung cancer and sarcoma (including intractable types of any of the above cancers), or one or more combinations of the above cancers. In one embodiment, in one embodiment, the cancer is colorectal cancer and optionally the chemotherapeutic agent is irinotecan. As used herein, "sarcoma" refers to the type of cancer that grows in connective tissue. Sarcomas are gastrointestinal stromal tumors (a type of soft tissue sarcoma found in the stomach and gastrointestinal tract, commonly known as GIST), soft tissue sarcomas (eg, smooth myoma, fibroblastic sarcoma, liposarcoma, capsicum). Includes sarcoma (KS), angiosarcoma, malignant peripheral nerve sheath tumor (MPNST), synovial sarcoma, horizontal print sarcoma) and osteosarcoma (eg chondrosarcoma, osteosarcoma, Ewing sarcoma, spinal cord tumor).

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody involved in the binding of the antibody to an antigen. Usually, the heavy and light chain variable domains of native antibodies (VH and VL, respectively) have similar structures, each containing four conserved framework regions (FR) and three hypervariable regions (HVR). Have. (E.g., Kindt et al., Kuby Immunology , 6 th ed., WH Freeman and Co., see page 91 to (2007).) To confer antigen-binding specificity, single VH or VL domains Is enough. Furthermore, an antibody that binds to a specific antigen is isolated using a VH domain or VL domain derived from an antibody that specifically binds to that antigen, and a library of each of the complementary VL domain or VH domain is screened. Can be done. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

As used herein, the term "antigen binding site of an antibody" refers to an amino acid residue of an antibody that is involved in antigen binding. The antigen-binding portion of an antibody comprises the amino acid residues of the "complementarity determining regions" or "CDRs". The "framework" or "FR" region is a variable domain region other than the hypervariable region residues defined herein. Thus, the variable domains of the light and heavy chains of an antibody include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. In particular, the heavy chain CDR3 is the region that contributes most to antigen binding and defines the properties of the antibody. The CDR and FR regions are determined by the standard definitions of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and / or " Those residues from the "super variable loop".

Antibody specificity refers to the selective recognition of an antibody for a particular epitope of an antigen. For example, natural antibodies are unispecific. The "bispecific antibody" according to the present invention is an antibody having two different antigen binding specificities. The antibodies of the invention are specific for two different antigens: DR5 as the first antigen and FAP as the second antigen.

As used herein, the term "unispecific" antibody means an antibody that has one or more binding sites, each of which binds to the same epitope of the same antigen.

As used herein, the term "bispecific" antibody means an antibody each having at least two binding sites that bind to the same antigen or different epitopes of different antigens.

The antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two different sites. Provided herein are bispecific antibodies with binding specificity to FAP and DR5. In certain embodiments, bispecific antibodies can bind to two different epitopes of DR5. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing DR5. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (Milstein and Cuello, Nature 305: 537 (1983), International. Publication No. 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)) and "knob-in-hole" engineering (see, eg, US Pat. No. 5,731,168). included. Multispecific antibodies also manipulate the electrostatic steering effect to create Fc-heterodimer molecules of the antibody (International Publication No. 2009/089004); crosslinking two or more antibodies or fragments ( See, eg, US Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); Using leucine zippers to generate bispecific antibodies (eg, Kostelny et al., J.). Immunol., 148 (5): 1547-1553 (1992)); using "diabody" techniques to generate bispecific antibody fragments (eg, Hollinger et al., Proc. Natl). Acad. Sci. USA, 90: 6444-6448 (1993)); using single chain Fv (sFv) dimers (eg, Gruber et al., J. Immunol., 152: 5368 (1994)). See); and can be made by preparing trispecific antibodies, eg, as described in Tutt et al. J. Immunol. 147: 60 (1991).

Manipulative antibodies having three or more functional antigen binding sites, including "Octopus antibodies", are also included herein (see, eg, US Patent Publication No. 2006/0025576).

Antibodies or fragments herein also include "Dual Acting", which comprises at least one antigen binding site that binds to FAP or DR5 and another different antigen (see, eg, US Patent Publication No. 2008/0069820). ) FAb ”or“ DAF ”is included.

As used herein, the term "valence" means the presence of a specific number of binding sites in an antibody molecule. Thus, the terms "divalent", "tetravalent" and "hexavalent" mean the presence of two binding sites, four binding sites and six binding sites, respectively, in the antibody molecule. Bispecific antibodies according to the invention are at least "divalent" and may be "trivalent" or "multivalent" (eg, "tetravalent" or "hexavalent").

The antibody of the present invention has two or more binding sites and is bispecific. That is, an antibody can also be bispecific if there are more than two binding sites (ie, the antibody is trivalent or multivalent). Bispecific antibodies of the invention include, for example, polyvalent single chain antibodies, diabodies and triabodies, and additional antigen binding sites (eg, single chain Fv, VH domain and / or VL domain, Fab, or (eg, single chain Fv, VH domain and / or VL domain, Fab, or (. Fab) 2) comprises an antibody having a constant domain structure of a full-length antibody to which one or more peptide linkers are bound. Antibodies can be full length from a single species, or can be chimeric or humanized.

As used herein, the term "amino acid" is used as alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), aspartic acid (asn, N), aspartic acid (asp, D). , Cystine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L) Leucine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine Means a group of naturally occurring carboxyα-amino acids, including (tyr, Y), and valine (val, V).

As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, the terms "transfectant" and "transfected cells" include primary target cells and cultures derived from them, regardless of the number of migrations. Also, not all progeny are exactly the same in DNA amount due to the effects of planned or unintended mutations. Includes mutant offspring with the same function or biological activity as screened in the original transformed cells.

"Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise stated, "binding affinity" refers to an essential binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). .. In general, the affinity of the molecule X for its partner Y is represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific descriptive and exemplary embodiments for measuring binding affinity are described below.

As used herein, the term "binding" or "specific binding" refers to an epitope of an antigen in an in vitro assay, preferably a surface plasmon resonance assay (SPR, BIAcore, GE-Healthcare Uppsala, Sweden). Refers to antibody binding. The affinity of binding is defined by the terms ka (rate constant for antibody association from antibody / antigen complex), kD (dissociation constant) and KD (kD / ka). The binding or specific binding has a binding affinity (KD) of ^10-8 mol / l or less, preferably ^10-9 M to ^{10-13 mol / l.}

Binding of the antibody to the desreceptor can be investigated by the BIAcore assay (GE-Healthcare Uppsala, Sweden). The affinity of binding is defined by the terms ka (rate constant for antibody association from antibody / antigen complex), kD (dissociation constant) and KD (kD / ka).

The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, the epitope determinant comprises a chemically active surface grouping of the molecule, such as an amino acid, sugar side chain, phosphoryl or sulfonyl, and in certain embodiments it is a particular three-dimensional structural feature. And / or may have specific charging characteristics. An epitope is a region of an antigen that is bound by an antibody.

As used herein, the terms "manipulate", "manipulated", "manipulate" and the term "glycosylation manipulator", particularly with the prefix "glyco-", are naturally occurring or recombinant. It is believed to include any manipulation of the glycosylation pattern of the polypeptide or fragment thereof. Glycosylation manipulations include metabolic manipulations of the cellular glycosylation mechanism, including genetic manipulation of oligosaccharide synthesis pathways to achieve changes in glycosylation of glycoproteins expressed in the cell. In addition, glycosylation manipulations include mutations in glycosylation and the effects of the cellular environment. In one embodiment, the glycosylation operation is a modification of glycosyltransferase activity. In certain embodiments, the manipulation results in a modification of glucosaminyltransferase activity and / or fucosyltransferase activity.

All separate embodiments may be combined.

［式中、
Ａは、

からなる群より選択され、
ｎは、０又は１であり、
Ｒ_１は、Ｈ、ハロゲン又はＣ_１−６−アルキルからなる群より選択され、
Ｒ_２は、Ｈ、ハロゲン又はＣ_１−６−アルキルからなる群より選択され、
Ｒ_３は、Ｈ、ハロゲン又はＣ_１−６−アルキルからなる群より選択され、
Ｒ_４は、Ｃ_１−６−アルキル、Ｃ_１−６−アルコキシ−Ｃ_１−６−アルキル又はＣ_３−６−ヘテロシクロアルキルからなる群より選択され、
Ｒ_５は、Ｈ、ハロゲン又はＣ_１−６−アルキルからなる群より選択され、
Ｒ_６は、Ｈ、ハロゲン又はＣ_１−６−アルキルからなる群より選択され、
Ｌは、

からなる群より選択され、
ｍは、１、２又は３であり、
ｋは、１、２又は３である］
の化合物又はその薬学的に許容される塩に関する。 In one embodiment, the present invention describes the formula (I):

[During the ceremony,
A is

Selected from the group consisting of
n is 0 or 1 and
R ₁ is selected from the group consisting of H, halogen or C _1-6-alkyl.
R ₂ is selected from the group consisting of H, halogen or C _1-6-alkyl.
R ₃ is selected from the group consisting of H, halogen or C _1-6-alkyl.
R _{4 is, C 1-6} - _{alkyl, C 1-6} - alkoxy _{-C 1-6} - alkyl or _{C 3-6} - is selected from the group consisting of heterocycloalkyl,
R ₅ is selected from the group consisting of H, halogen or C _1-6-alkyl.
R ₆ is selected from the group consisting of H, halogen or C _1-6-alkyl.
L is

Selected from the group consisting of
m is 1, 2 or 3
k is 1, 2 or 3]
With respect to the compound of or a pharmaceutically acceptable salt thereof.

［式中、ｍは１、２又は３、且つｋは１、２又は３である。］
からなる群より選択される。 One embodiment of the present invention relates to a compound of formula I described herein, wherein the LA group is:

[In the formula, m is 1, 2 or 3, and k is 1, 2 or 3. ]
Selected from the group consisting of.

One embodiment of the invention relates to a compound of formula I described herein _{, wherein R 1 is F.}

One embodiment of the present invention relates to a compound of formula I described herein, wherein A is A-1.

One embodiment of the present invention relates to a compound of formula I described herein, wherein A is A-1 and n is 0.

One embodiment of the present invention relates to a compound of formula I described herein, wherein A is A-1 and n is 1.

One embodiment of the present invention relates to a compound of formula I described herein _{, wherein A is A-1 and R 2} and R _{3 are F.}

からなる群より選択される、本明細書に記載の式Ｉの化合物に関する。 In one embodiment of the present invention, A is A-1, n is 0, R ₂ and R ₃ are F, and R ₄ is.

It relates to a compound of formula I described herein, selected from the group consisting of.

からなる群より選択される、本明細書に記載の式Ｉの化合物に関する。 In one embodiment of the present invention, A is A-1, n is 1, R ₂ and R ₃ are F, and R ₄ is.

It relates to a compound of formula I described herein, selected from the group consisting of.

One embodiment of the present invention relates to a compound of formula I described herein, wherein A is A-2.

One embodiment of the invention, A is an A-2, and either R ₅ is F, or A is A-4, and R ₆ is F, a compound of formula I.

One embodiment of the invention, A is an A-2, R ₅ is F, a compound of formula I as described herein.

One embodiment of the invention relates to a compound of formula I described herein, wherein A is A-3.

One embodiment of the invention, A is an A-4, R ₆ is F, a compound of formula I as described herein.

One embodiment of the invention relates to a compound of formula I described herein, wherein L is L-1, L-2 or L-3.

One embodiment of the present invention relates to a compound of formula I described herein, wherein L is L-1.

One embodiment of the present invention relates to a compound of formula I described herein, wherein L is L-2.

One embodiment of the present invention relates to a compound of formula I described herein, wherein L is L-2 and m is 1.

One embodiment of the present invention relates to a compound of formula I described herein, wherein L is L-2 and m is 2.

One embodiment of the present invention relates to a compound of formula I described herein, wherein L is L-2 and m is 3.

One embodiment of the present invention relates to a compound of formula I described herein, wherein L is L-3.

One embodiment of the invention relates to a compound of formula I described herein, wherein L is L-4 or L-6.

One embodiment of the present invention relates to a compound of formula I described herein, wherein L is L-4.

One embodiment of the present invention relates to a compound of formula I described herein, wherein L is L-5.

One embodiment of the present invention relates to a compound of formula I described herein, wherein L is L-5 and k is 3.

One embodiment of the present invention relates to a compound of formula I described herein, wherein L is L-6.

One embodiment of the present invention is
N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-) 2H-pyran-4-yl) acetyl) isoindolin-5-yl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-methyl-2- ( ((R) -3-Methylmorpholino) Methyl) Piperazin-1-yl) Acetyl) Indolin-3-Carboxamide Trihydrochloride,
(3R) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino)) Propanoyl] amino] -2- (oxan-4-yl) acetyl] -1,3-dihydroisoindole-5-yl] amino] -2-oxoethyl] -6-[(4-fluorophenyl) methyl] -3 -Methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazin-1-yl] acetyl] -2H-indole -3-Carboxamide trihydrochloride,
(3S) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino)) Propanoyl] amino] -2- (oxan-4-yl) acetyl] -1,3-dihydroindole-5-yl] amino] -2-oxoethyl] -6-[(4-fluorophenyl) methyl] -3- Methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazine-1-yl] acetyl] -2H-indole- 3-Carboxamide trihydrochloride,
(3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2- (methylamino) propanoyl] amino] butanoyl] -7-[[2-[[(3S)- 6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] ] Piperazin-1-yl] acetyl] -2H-indole-3-carbonyl] amino] acetyl] amino] -N-[(1R) -1,2,3,4-tetrahydronaphthalene-1-yl] -3, 4-Dihydro-1H-isoquinolin-3-carboxamide trihydrochloride,
(3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2- (methylamino) propanoyl] amino] butanoyl] -7-[[2-[[(3R)- 6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] ] Piperazin-1-yl] acetyl] -2H-indole-3-carbonyl] amino] acetyl] amino] -N-[(1R) -1,2,3,4-tetrahydronaphthalene-1-yl] -3, 4-Dihydro-1H-isoquinolin-3-carboxamide trihydrochloride,
(3S) -2-((S) -3,3-dimethyl-2-((S) -2- (methylamino) propanoamide) butanoyl) -7- (6- (4-fluorobenzyl) -3-3 Methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) -N- ((R) -1,2,3,4-tetrahydronaphthalene-1-yl) -1,2,3,4-tetrahydroisoquinolin-3-carboxamide trihydrochloride,
(3S) -2-((S) -3,3-dimethyl-2-((S) -2- (methylamino) propanoamide) butanoyl) -7- (2- (6- (4-fluorobenzyl)) -3-Methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) Acetamide) -N-((R) -1,2,3,4-tetrahydronaphthalene-1-yl) -1,2,3,4-tetrahydroisoquinolin-3-carboxamide trihydrochloride,
(3S) -N- (2,6-difluorophenyl) -7- (2- (6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-methyl-) 2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) acetamide) -2-((S) -2-((S) -2- (methyl) Amino) Propanamide) -2- (Tetrahydro-2H-Pyran-4-yl) Acetyl) -1,2,3,4-Tetrahydroisoquinolin-3-carboxamide Trihydrochloride,
(R) -N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl) -2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(R, R, R) -N, N'-(ethane-1,2-diyl) bis (6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5) -Methyl-2-(((R) -3-methylmorpholino) methyl) piperazine-1-yl) acetyl) indoline-3-carboxamide) trihydrochloride,
(S) -N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl) -2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide,
(S) -N- (2-(((S) -1-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propane) Amide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2) -((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((2S, 3R) -3-methoxy-2-((S) -2) -(Methylamino) propanamide) butanoyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)-)- 5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((2S, 3S) -3-methyl-2-((S) -2) -(Methylamino) propanamide) pentanoyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)-)- 5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -3,3-dimethyl-2-((S) -2) -(Methylamino) propanamide) butanoyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)-)- 5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(S) -N- (3-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propane) Amide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-5-yl) amino) -3-oxopropyl) -6- (4-fluorobenzyl) -3-methyl-1- ( 2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(S) -N- (4-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propane) Amide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-5-yl) amino) -4-oxobutyl) -6- (4-fluorobenzyl) -3-methyl-1- (2) -((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
N-(2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R) , 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
N- (2- (4-Fluoro-3-((S) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-) 4-yl) Acetyl) Isoindrin-1-Carboxamide) Benzamide) Ethyl) -6- (4-Fluorobenzyl) -3-Methyl-1-(2-((2R, 5R) -5-Methyl-2- ( ((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide dihydrochloride,
N-(4-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) carbamoyl) phenyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)) -5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
N- (4- (4-Fluoro-3-((S) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-) 4-Il) Acetyl) Isoindrin-1-Carboxamide) Benzamide) Phenyl) -6- (4-Fluorobenzyl) -3-Methyl-1-(2-((2R, 5R) -5-Methyl-2- ( ((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride, and N1-((S) -3-((2,6-difluorophenyl) carbamoyl)) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) -N3- (3-(6- (4-Fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-) 1-Il) Acetyl) Indoline-3-yl) propyl) Malonamide With respect to the compounds described herein selected from the group consisting of trihydrochloride.

One embodiment of the invention relates to the compounds described herein for use as therapeutically active substances.

One embodiment of the invention relates to the compounds described herein for use in therapeutic and / or prophylactic treatment of cancer.

［式中、
Ｒ_１は、Ｈ、ハロゲン又はＣ_１−６−アルキルからなる群より選択され、
Ｒ４は、

からなる群より選択される］
の二量体化合物の式Ｉ−ａのモノマーに関する。 One embodiment of the present invention is the formula I.

[During the ceremony,
R ₁ is selected from the group consisting of H, halogen or C _1-6-alkyl.
R4 is

Selected from the group consisting of]
With respect to the monomer of formula Ia of the dimeric compound of.

One embodiment of the present invention relates to a pharmaceutical composition comprising any of the compounds described herein, or a pharmaceutically acceptable salt thereof, as an active ingredient, together with a pharmaceutically acceptable carrier or additive. ..

One embodiment of the invention relates to a compound of formula I described herein for use as a therapeutically active substance.

One embodiment of the invention relates to a compound of formula I described herein for use in therapeutic and / or prophylactic treatment of cancer.

One embodiment of the present invention as inhibitors of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or function as inhibitors of active caspase proteins that bind to I AP, cancer, in particular solid tumors With respect to compounds for use as a combination therapy with a bispecific antibody in a method of improvement, treatment or control, the bispecific antibody is here the death receptor 5 (DR5) and a fibroblast activation protein ( It binds to FAP) and comprises at least one DR5-specific antigen-binding site and at least one FAP-specific antigen-binding site.

One embodiment of the invention binds to an apoptosis inhibitor protein (IAP) in combination with the desreceptor 5 (DR5) described herein and a bispecific antibody that binds to a fibroblast activation protein (FAP). as inhibitors of SMAC proteins, and / or to compounds of formula I according to any one of claims 1 to 8 which functions as an inhibitor of active caspase proteins that bind to I AP.

One embodiment of the present invention as inhibitors of SMAC which bind to inhibitor of apoptosis protein (IAP), and / or function as inhibitors of active caspase proteins that bind to I AP, double described herein With respect to a compound for use as a combination therapy with a specific antibody , where the bispecific antibody is a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8. At least one DR5-specific antigen binding site containing a region and at least one FAP-specific containing a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 16. Includes antigen binding sites of.

One embodiment of the invention binds to an apoptosis inhibitor protein (IAP) in combination with a bispecific antibody that binds to the death receptor 5 (DR5) and fibroblast activation protein (FAP) described herein. as inhibitors of SMAC to, and / or relates to compounds of formula I which acts as an inhibitor of activated caspase proteins that bind to I AP, wherein the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 7 At least one DR5-specific antigen binding site containing the variable heavy chain region and the variable light chain region containing the amino acid sequence of SEQ ID NO: 8, and the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and the amino acid of SEQ ID NO: 16. It comprises at least one FAP-specific antigen binding site comprising a light chain variable region comprising a sequence.

One embodiment of the present invention as inhibitors of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or function as an inhibitor of activated caspase proteins that bind to I AP, cancer, in particular solid tumors For use as a combination therapy with a bispecific antibody that binds to death receptor 5 (DR5) and fibroblast activation protein (FAP) described herein in a method of ameliorating, treating or controlling. With respect to the compounds, the cancers here are rectal colon cancer, sarcoma, head and neck cancer, squamous cell carcinoma, breast cancer, pancreatic cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer and mesenteric tumor. be.

One embodiment of the present invention as inhibitors of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or function as an inhibitor of activated caspase proteins that bind to I AP, cancer, in particular solid tumors For use as a combination therapy with a bispecific antibody that binds to desreceptor 5 (DR5) and fibroblast activation protein (FAP) described herein in a method of ameliorating, treating or controlling. With respect to the compound, the SMAC protein inhibitor and bispecific antibody that bind to the apoptosis inhibitor protein (IAP) are administered alternately.

One embodiment of the invention binds to an apoptosis inhibitor protein (IAP) in combination with a bispecific antibody that binds to the desreceptor 5 (DR5) and fibroblast activation protein (FAP) described herein. as inhibitors of SMAC proteins, and / or, with respect to compounds of formula I as described herein that function as inhibitors of activated caspase proteins that bind to I AP, wherein the bispecific antibody has the sequence The amino acid sequences of Nos. 18, 19 and 20 are included, or the bispecific antibody comprises the amino acid sequences of SEQ ID NOs: 17, 19 and 20.

One embodiment of the invention binds to an apoptosis inhibitor protein (IAP) in combination with a bispecific antibody that binds to the desreceptor 5 (DR5) and fibroblast activation protein (FAP) described herein. as inhibitors of SMAC proteins, and / or, with respect to compounds of formula I as described herein that function as inhibitors of activated caspase proteins that bind to I AP, wherein the bispecific antibody has the sequence The amino acid sequences of Nos. 18, 19 and 20 are included, or the bispecific antibody comprises the amino acid sequences of SEQ ID NOs: 17, 19 and 20.

One embodiment of the present invention as inhibitors of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or function as an inhibitor of activated caspase proteins that bind to I AP, cancer, in particular solid tumors For use as a combination therapy with a bispecific antibody that binds to desreceptor 5 (DR5) and fibroblast activation protein (FAP) described herein in a method of ameliorating, treating or controlling. With respect to the compound, the antibody here is human.

One embodiment of the present invention as inhibitors of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or function as an inhibitor of activated caspase proteins that bind to I AP, cancer, in particular solid tumors For use as a combination therapy with a bispecific antibody that binds to desreceptor 5 (DR5) and fibroblast activation protein (FAP) described herein in a method of ameliorating, treating or controlling. With respect to the compound, here the antibody is humanized.

One embodiment of the present invention as inhibitors of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or function as an inhibitor of activated caspase proteins that bind to I AP, cancer, in particular solid tumors For use as a combination therapy with a bispecific antibody that binds to desreceptor 5 (DR5) and fibroblast activation protein (FAP) described herein in a method of ameliorating, treating or controlling. With respect to a pharmaceutical composition comprising a compound.

One embodiment of the present invention as inhibitors of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or function as an inhibitor of activated caspase proteins that bind to I AP, use as a combination therapy To the death receptor 5 (DR5) and fibroblast activation protein (FAP) described herein in a first container containing a compound for and in a method of ameliorating, treating or controlling cancer, especially solid tumors. The present invention relates to a kit comprising a second container containing a bispecific antibody that binds.

One embodiment of the present invention as inhibitors of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or, the compound herein that function as inhibitors of activated caspase proteins that bind to the I AP The use of bispecific antibodies that bind to desreceptor 5 (DR5) and fibroblast activation protein (FAP) as described in methods of ameliorating, treating or controlling cancers, especially solid tumors .

One embodiment of the present invention relates to the use of a compound described herein or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the therapeutic and / or prophylactic treatment of cancer.

One embodiment of the invention induces apoptosis of target cells with respect to bispecific antibodies that bind to FAPs and DR5s that specifically crosslink the death receptor. The advantage of these bispecific death receptor agonist antibodies over common death receptors that target the antibody is the specificity of inducing apoptosis only at the site where FAP is expressed. As outlined above, the inventors of the present invention have developed DR5 binding moieties that have superior properties compared to known DR5 binding agents that can be incorporated into novel advantageous DR5-FAP bispecific antibodies. bottom.

One embodiment of the present invention is
(A) Heavy chain CDR1 of SEQ ID NO: 1;
(B) Heavy chain CDR2 of SEQ ID NO: 2;
(C) Heavy chain CDR3 of SEQ ID NO: 3;
(D) Light chain CDR1 of SEQ ID NO: 4;
(E) Light chain CDR2 of SEQ ID NO: 5;
(F) Light chain CDR3 of SEQ ID NO: 6;
With at least one DR5-specific antigen binding site, including
a) Heavy chain CDR1 of SEQ ID NO: 9;
(B) Heavy chain CDR2 of SEQ ID NO: 10;
(C) Heavy chain CDR3 of SEQ ID NO: 11;
(D) Light chain CDR1 of SEQ ID NO: 12;
(E) Light chain CDR2 of SEQ ID NO: 13;
(F) Light chain CDR3 of SEQ ID NO: 14.
With respect to a therapeutic combination comprising a bispecific antibody that binds to DR5 and FAP, comprising at least one FAP-specific antigen binding site.

A first embodiment of the present invention comprises at least one DR5-specific antigen binding site comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 8; A bispecific antibody comprising at least one FAP-specific antigen binding site comprising a heavy chain variable region comprising the amino acid sequence of No. 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

One embodiment of the invention relates to bispecific antibodies comprising SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

One embodiment of the invention relates to bispecific antibodies comprising SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 20.

In one embodiment of the invention, bispecific antibodies that bind DR5 and FAP are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, with the amino acid sequence of SEQ ID NO: 7. With at least one DR5-specific antigen binding site containing a heavy chain variable domain (VH) with 97%, 98%, 99% or 100% sequence identity; the variable heavy chain of SEQ ID NO: 15 and SEQ ID NO: 16 Includes at least one FAP-specific antigen binding site, including the variable light chain of.

One embodiment of the invention relates to a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Bispecific antibodies that bind to DR5 and FAP, including substitutions (eg, conservative substitutions), insertions, or deletions with respect to the reference sequence, retain the ability to bind to FAP and DR5. .. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 7. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (ie, within the FR). In some cases, bispecific antibodies that bind DR5 and FAP include the VH sequence of SEQ ID NO: 7 (including post-translational modifications of that sequence).

In one embodiment of the invention, bispecific antibodies that bind DR5 and FAP are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, with the amino acid sequence of SEQ ID NO: 8. With at least one DR5-specific antigen binding site containing a light chain variable domain (VL) with 97%, 98%, 99% or 100% sequence identity; the variable heavy chain of SEQ ID NO: 15 and SEQ ID NO: 16 Includes at least one FAP-specific antigen binding site, including the variable light chain of.

One embodiment of the invention relates to a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Bispecific antibodies that bind to DR5 and FAP, including substitutions (eg, conservative substitutions), insertions, or deletions with respect to the reference sequence, retain the ability to bind to DR5 and FAP. .. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (ie, within the FR). In some cases, bispecific antibodies that bind DR5 and FAP include the VL sequence of SEQ ID NO: 8 (including post-translational modifications of that sequence).

One embodiment of the invention comprises at least one DR5-specific antigen binding site comprising a variable light chain of SEQ ID NO: 8 and a variable heavy chain of SEQ ID NO: 7; and at least 90% of the amino acid sequence of SEQ ID NO: 15. FAP-specific, including heavy chain variable domain (VH) sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. With respect to a bispecific antibody that binds to DR5 and FAP, including at least one antigen binding site. In certain embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity are relative to reference sequences. Bispecific antibodies that bind to DR5 and FAP, including substitutions (eg, conservative substitutions), insertions, or deletions, retain the ability to bind to FAP and DR5. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 15. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (ie, within the FR). In some cases, bispecific antibodies that bind DR5 and FAP include the VH sequence of SEQ ID NO: 15 (including post-translational modifications of that sequence).

One embodiment of the invention comprises at least one DR5-specific antigen binding site comprising a variable light chain of SEQ ID NO: 8 and a variable heavy chain of SEQ ID NO: 7; and at least 90% of the amino acid sequence of SEQ ID NO: 16. FAP-specific, including light chain variable domain (VL) sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. With respect to a bispecific antibody that binds to DR5 and FAP, including at least one antigen binding site. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is relative to a reference sequence. Bispecific antibodies that bind to DR5 and FAP, including substitutions (eg, conservative substitutions), insertions, or deletions, that contain the sequence retain the ability to bind to DR5 and FAP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 16. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVR (ie, within the FR). In some cases, bispecific antibodies that bind DR5 and FAP include the VL sequence of SEQ ID NO: 16 (including post-translational modifications of that sequence).

One embodiment of the invention relates to bispecificity that binds to DR5 and FAP, where the antibody is similar to any of the VHs shown above, and any of the embodiments shown above. Includes VL. In one embodiment, the antibody comprises the VH sequences of SEQ ID NOs: 7 and 8 and the VL sequences of SEQ ID NOs: 15 and 16 (including post-translational modifications of the sequences).

In one embodiment of the invention, the bispecific antibody that binds to DR5 and FAP according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody or a human antibody. In one embodiment, the bispecific antibody that binds DR5 and FAP according to any of the above embodiments is a human antibody.

Compounds of formula I and salts thereof may contain at least one asymmetric carbon atom and thus exist as a mixture of different stereoisomers. The various isomers can be isolated by known separation methods, such as chromatography.

The compounds disclosed herein and included in Formula I above may exhibit tautomerism or structural isomerism. The present invention includes all tautomeric or structurally heteromorphic forms of these compounds or mixtures of such forms and is not limited to any one of the tautomeric or structurally heterogeneous forms represented by the above formulas.

arrangement

Dosage The compounds of the invention preferably bind to the BIR domain of IAP and prevent IAP from binding to other proteins. Examples of BIR-binding proteins include, but are not limited to, caspase 3, caspase 7, caspase 9, Smac and the like. Examples of IAPs include, but are not limited to, XIAP, cIAP1, cIAP2 or NAIP. In one aspect, the compounds of the invention bind to the BIR2 and / or BIR3 domains of XIAP, cIAP1, and / or cIAP2. In another aspect, the compounds of the invention bind to the BIR2 domain of XIAP, cIAP1, and / or cIAP2.

The compounds of the present invention are useful, especially in cancer cells, for inducing apoptosis in cells or for sensitizing cells to apoptosis signals. Apoptotic signals can be induced in cancer cells, for example by radiation therapy or antitumor chemotherapy. Alternatively, the apoptotic signal can be induced in cancer cells by activating the death receptor with a death receptor agonist. Desceptor agonists are either naturally occurring, such as tumor necrosis factor α (TNF-α), or non-naturally occurring, such as synthetic antibodies such as DR4 or DR5 antibodies.

Therefore, the compounds of the present invention are particularly useful for ameliorating, controlling or treating cell proliferation disorders such as oncological disorders. Such compounds and preparations containing the compounds are expected to be useful for the treatment or control of blood cancers such as acute myeloid leukemia or solid tumors such as tumors of breast, colon, lung and prostate. Has been done.

The "therapeutically effective amount" or "effective amount" of a compound according to the present invention means an amount of a compound that is effective for preventing, alleviating or ameliorating the symptoms of a disease or for prolonging the life of a subject to be treated. The determination of a therapeutically effective amount is within the scope of the art.

The therapeutically effective or dosage of the compounds according to the invention will vary over a wide range and may be determined in a manner known in the art. Such doses are tailored to the individual requirements of each individual case, including the particular compound being administered, the route of administration, the condition being treated, and the patient being treated. For oral or parenteral administration to an adult weighing about 70 kg, a daily dose of about 10 mg to about 10000 mg, preferably about 200 mg to about 1000 mg is generally appropriate, but may exceed the upper limit if instructed. .. The daily dose can be administered as a single dose or in divided doses, or in the case of parenteral administration, it may be administered as one or more bolus injections or as a continuous injection.

Drugs useful in the practice of the invention, ie, drugs containing the compounds of the invention, are internally, eg, orally (eg, tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions or suspensions. It can be administered nasally (eg, in the form of nasal drops) or rectally (eg, in the form of suppositories). However, administration can also be done parenterally, eg, intramuscularly or intravenously (eg, in the form of an injection). In addition, administration can be done topically (eg in the form of ointments, creams or oils).

Composition / Formulation In an alternative embodiment, the invention is a pharmaceutical formulation comprising at least one compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable additive and / or carrier. Contains the composition.

These pharmaceutical compositions can be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulation can be conveniently provided in unit dosage form and can be prepared by any method well known in the field of pharmacy. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. Generally, the amount of active ingredient that can be combined with the carrier material to produce a single dosage form is the amount of compound of formula I that produces a therapeutic effect. The amount of such active ingredient will typically range from about 1 percent to about 99 percent of 100 percent, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent. ..

A method of preparing such a formulation or composition comprises the step of associating the compound of the invention with a carrier and optionally one or more sub-ingredients. Generally, a pharmaceutical product is prepared by uniformly and closely associating the compound of the present invention with a liquid carrier and / or a fine powder solid support, and then molding the product as required.

The compounds of formula I and their pharmaceutically acceptable salts and esters shall be treated with a pharmaceutically inert inorganic or organic adjuvant for the production of tablets, coated tablets, sugar-coated tablets, and hard gelatin capsules. Can be done. Lactose, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, microcrystalline cellulose, corn starch or derivatives thereof, talc, stearic acid or salts thereof, etc., as described above, for example for tablets, sugar-coated tablets, and hard gelatin capsules. Can be used as an adjuvant.

Suitable adjuvants for soft gelatin capsules are, for example, vegetable oils, waxes, fats and oils, semi-solid substances and liquid polyols. Adjuvants suitable for the manufacture of solutions and syrups are, for example, H _{2 O,} polyols, sucrose, invert sugar, glucose and the like. Adjuvants suitable for injection solutions are, for example, H _{2 O,} alcohols, polyols, glycerol, vegetable oil and the like. Suitable adjuvants for suppositories are, for example, natural oils or hydrogenated oils, waxes, fats and oils, semi-solid polyols, liquid polyols and the like. Suitable adjuvants for topical preparations are glycerides, semi-synthetic and synthetic glycerides, hydrogenated oils, liquid waxes, liquid paraffins, liquid fatty alcohols, sterols, polyethylene glycols and cellulose derivatives.

Moreover, pharmaceutical preparations include preservatives, solubilizers, thickeners, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavors, salts for altering osmotic pressure, buffers, masking agents or antioxidants. It can contain oxidizing substances. Pharmaceutical preparations can also contain other therapeutic substances.

The compound in the present invention (compound of general formula I) can be prepared by the general reaction scheme specified in the following scheme.

工程Ａ：６０℃から１２０℃の適切な溶媒、例えばジオキサン、テトラヒドロフラン、ジメトキシエタン又はジグリム中パラジウム触媒、例えば［１，１−ビス（ジフェニルホスフィノ）フェロセン］ジクロロパラジウム（ＩＩ）又は［１，１−ビス（ジフェニルホスフィノ）フェロセン］ジクロロパラジウム（ＩＩ）ジクロロメタン錯体を１時間から２４時間使用することにより、ブロモ誘導体ＩＩと適切な有機金属試薬、例えば（４−フルオロベンジル）塩化亜鉛（ＩＩ）をカップリングして、誘導体ＩＩＩを形成することを達成することができる。
特定の条件は、シールド管内のテトラヒドロフラン中［１，１−ビス（ジフェニルホスフィノ）フェロセン］ジクロロパラジウム（ＩＩ）ジクロロメタン錯体を９０℃で１８時間使用することである。

Step A: Palladium catalyst in a suitable solvent from 60 ° C to 120 ° C, such as dioxane, tetrahydrofuran, dimethoxyethane or diglycim, such as [1,1-bis (diphenylphosphino) ferrocene] dichloropalladium (II) or [1,1 -Bis (diphenylphosphino) ferrocene] Dichloropalladium (II) dichloromethane complex is used for 1 to 24 hours to obtain a bromo derivative II and a suitable organometallic solvent such as (4-fluorobenzyl) zinc (II) chloride. Coupling can be achieved to form derivative III.
A specific condition is to use the [1,1-bis (diphenylphosphino) ferrocene] dichloropalladium (II) dichloromethane complex in tetrahydrofuran in a shielded tube at 90 ° C. for 18 hours.

Step B :: Cleavage of the amino protecting group from Derivative III can be performed by various methods known in the art. The tert-butoxycarbonyl group is a mineral acid such as trifluoroacetic acid, dichloroacetic acid, acetic acid or p-toluenesulfonic acid in a solvent such as dichloromethane, chloroform, tetrahydrofuran, methanol, ethanol or water at 0-60 ° C. The particular protecting group PG that can be cleaved is the tert-butoxycarbonyl group. A preferred condition is to use HCl in dioxane at 50 ° C. for 5 hours.

Step C: The amide coupling is performed by amide coupling reagents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), O- (benzotriazole-1-yl) -N. , N, N', N'-tetramethyluronium-hexafluorophosphate (HBTU), (1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate (HATU), O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium-tetrafluoroborate (TBTU), propylphosphonic acid anhydride (TBTU) T3P) etc. and amine IV and acid V in the presence of solvents such as methylene chloride, 1,2-dichloroethane, tetrahydrofuran, dimethylformamide or ethyl acetate bases such as triethylamine, diisopropylethylamine, N-methylmorpholin or pyridine It can be achieved by the reaction with. Specific conditions are the use of propylphosphone anhydride and N-methylmorpholine in ethyl acetate for 18 hours at room temperature.

Step D: Saponification of ester VI is accomplished by treatment with a base such as lithium hydroxide, potassium hydroxide or sodium hydroxide in water or a mixture of water and an organic solvent such as tetrahydrofuran, ethanol or methanol. Can be done. A specific condition is the use of lithium hydroxide in a mixture of ethanol and water at 60 ° C. for 3 hours.

Step E: The amide coupling is performed by amide coupling reagents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), O- (benzotriazole-1-yl) -N. , N, N', N'-tetramethyluronium-hexafluorophosphate (HBTU), (1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate (HATU), O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium-tetrafluoroborate (TBTU), propylphosphonic acid anhydride (TBTU) T3P) etc. and the acid VIIamine VIII in the presence of a solvent such as methylene chloride, 1,2-dichloroethane, tetrahydrofuran, dimethylformamide or a base in ethyl acetate, such as triethylamine, diisopropylethylamine, N-methylmorpholine or pyridine. It can be achieved by reaction. Specific conditions are the use of propylphosphone anhydride and N-methylmorpholine in ethyl acetate for 18 hours at room temperature.

Step F: Removal of Boc N-protecting groups involves mineral acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or solvents at 0 ° C to 80 ° C, such as dichloromethane, chloroform, tetrahydrofuran, methanol, ethanol or organic acids in water, such as tri. This can be done with fluoroacetic acid, dichloroacetic acid, acetic acid or p-toluenesulfonic acid. A specific condition is the use of HCl in a mixture of dioxane and ethyl acetate for 18 hours at room temperature.

Step G: The amide coupling is performed by amide coupling reagents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), O- (benzotriazole-1-yl) -N. , N, N', N'-tetramethyluronium-hexafluorophosphate (HBTU), (1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate (HATU), O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium-tetrafluoroborate (TBTU), propylphosphonic acid anhydride (TBTU) T3P) etc. and acids in the presence of solvents such as methylene chloride, 1,2-dichloroethane, tetrahydrofuran, dimethylformamide or ethyl acetate bases such as triethylamine, diisopropylethylamine, N-methylmorpholin or pyridine X Can be achieved by the reaction of. Specific conditions are the use of HATU and N-methylmorpholine in ethyl acetate for 18 hours at room temperature.

Step H: Saponification of ester XI is accomplished by treatment with a base such as lithium hydroxide, potassium hydroxide or sodium hydroxide in water or a mixture of water and an organic solvent such as tetrahydrofuran, ethanol or methanol. Can be done. A specific condition is the use of lithium hydroxide in a mixture of ethanol and water at 60 ° C. for 3 hours.

Step I: The amide coupling is performed by amide coupling reagents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), O- (benzotriazole-1-yl) -N. , N, N', N'-tetramethyluronium-hexafluorophosphate (HBTU), O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium-tetrafluoroborate In the presence of (TBTU), propylphosphonic acid anhydride (T3P), etc. and solvents such as methylene chloride, 1,2-dichloroethane, tetrahydrofuran or ethyl acetate medium bases such as triethylamine, diisopropylethylamine, N-methylmorpholine or pyridine. It can be achieved by the reaction of acid XII amine VIII in. Specific conditions are the use of propylphosphone anhydride and N-methylmorpholine in ethyl acetate for 18 hours at room temperature.

Step J: Removal of Boc N-protecting groups involves mineral acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or solvents at 0 ° C to 80 ° C, such as dichloromethane, chloroform, tetrahydrofuran, methanol, ethanol or organic acids in water, such as tri. This can be done with fluoroacetic acid, dichloroacetic acid, acetic acid or p-toluenesulfonic acid. A specific condition is the use of HCl in a mixture of dioxane and ethyl acetate for 18 hours at room temperature.

エステルＩＶａは、キラル分離により、その光学異性体形態ＩＶａ−１とＩＶａ−２に分離することができる。好ましいキラル分離は、キラル固定相、例えばＣｈｉｒａｌｐａｋ ＡＤ、及び適切な溶媒、例えばイソプロパノールとヘプタンの混合物を使用するキラルクロマトグラフィーである。

Ester IVa can be separated into its optical isomer forms IVa-1 and IVa-2 by chiral separation. A preferred chiral separation is chiral chromatography using a chiral stationary phase, such as Chiralpac AD, and a suitable solvent, such as a mixture of isopropanol and heptane.

工程Ａ：６０℃から１２０℃の適切な溶媒、例えばジオキサン、テトラヒドロフラン、ジメトキシエタン又はジグリム中パラジウム触媒、例えば［１，１−ビス（ジフェニルホスフィノ）フェロセン］ジクロロパラジウム（ＩＩ）又は［１，１−ビス（ジフェニルホスフィノ）フェロセン］ジクロロパラジウム（ＩＩ）ジクロロメタン錯体を１時間から２４時間使用することにより、ブロモ誘導体ＸＩＶと適切な有機金属試薬、例えば（４−フルオロベンジル）塩化亜鉛（ＩＩ）をカップリングして、誘導体ＸＶを形成することを達成することができる。
特定の条件は、シールド管内のテトラヒドロフラン中［１，１−ビス（ジフェニルホスフィノ）フェロセン］ ジクロロパラジウム（ＩＩ）ジクロロメタン錯体を９０℃で２時間使用することである。

Step A: Palladium catalyst in a suitable solvent from 60 ° C to 120 ° C, such as dioxane, tetrahydrofuran, dimethoxyethane or diglycim, such as [1,1-bis (diphenylphosphino) ferrocene] dichloropalladium (II) or [1,1 -Bis (diphenylphosphino) ferrocene] Dichloropalladium (II) dichloromethane complex is used for 1 to 24 hours to obtain the bromo derivative XIV and a suitable organometallic solvent such as (4-fluorobenzyl) zinc (II) chloride. Coupling can be achieved to form derivative XV.
A specific condition is to use the [1,1-bis (diphenylphosphino) ferrocene] dichloropalladium (II) dichloromethane complex in tetrahydrofuran in a shielded tube at 90 ° C. for 2 hours.

Step B: Cleavage of the amino protecting group from the derivative XV can be performed by various methods known in the art. The tert-butoxycarbonyl group is a mineral acid such as trifluoroacetic acid, dichloroacetic acid, acetic acid or p-toluenesulfonic acid in a solvent such as dichloromethane, chloroform, tetrahydrofuran, methanol, ethanol or water at 0-60 ° C. The particular protecting group PG that can be cleaved is the tert-butoxycarbonyl group. A preferred condition is to use HCl in dioxane at 50 ° C. for 2 hours.

Step C: The amide coupling is performed by amide coupling reagents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), O- (benzotriazole-1-yl) -N. , N, N', N'-tetramethyluronium-hexafluorophosphate (HBTU), (1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate (HATU), O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium-tetrafluoroborate (TBTU), propylphosphonic acid anhydride (TBTU) T3P) etc. and amine XVI and acid V in the presence of solvents such as methylene chloride, 1,2-dichloroethane, tetrahydrofuran, dimethylformamide or ethyl acetate bases such as triethylamine, diisopropylethylamine, N-methylmorpholin or pyridine It can be achieved by the reaction with. Specific conditions are the use of HATU and diisopropylethylamine in dimethylformamide at room temperature for 18 hours.

Step D: Nitrile reduction is suitable in a solvent such as methanol or ethanol by reaction with a boron hydride reagent in the presence of a nickel salt, or with or without addition of ammonia at room temperature or high temperature. This can be achieved by using a different catalyst, such as Raney nickel. Specific conditions are the use of atmospheric pressure hydrogen, Raney nickel as a catalyst and a 7N ammonia solution in methanol as a solvent at 60 ° C. for 18 hours.

Step E: The amide coupling of amine XVIII with monobenzylmaloate is performed by amide coupling reagents such as dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), O- ( Bentotriazole-1-yl) -N, N, N', N'-tetramethyluronium-hexafluorophosphate (HBTU), (1- [bis (dimethylamino) methylene] -1H-1,2,3- Triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate) (HATU), O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium-tetrafluoroborate ( TBTU), propylphosphonic acid anhydride (T3P), etc., and solvents such as methylene chloride, 1,2-dichloroethane, tetrahydrofuran, dimethylformamide or ethyl acetate medium bases such as triethylamine, diisopropylethylamine, N-methylmorpholine or pyridine. Can be achieved in the presence. Specific conditions are the use of HATU and diisopropylethylamine in dimethylformamide at room temperature for 18 hours.

Step F: The saponification of ester XX is carried out by treatment with water or a base such as potassium hydroxide or sodium hydroxide in a mixture of water and an organic solvent such as tetrahydrofuran, ethanol or methanol, or at standard pressure. Alternatively, it can be achieved by hydrogenation with a solvent in solvent such as methanol or ethanol under increasing pressure, such as Pd-C. A specific condition is the use of a compound dissolved in hydrogen at atmospheric pressure, palladium on carbon as a catalyst and methanol at room temperature for 6 hours.

Step G: The amide coupling is performed by amide coupling reagents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), O- (benzotriazole-1-yl) -N. , N, N', N'-tetramethyluronium-hexafluorophosphate (HBTU), O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium-tetrafluoroborate In the presence of (TBTU), propylphosphonic acid anhydride (T3P), etc. and solvents such as methylene chloride, 1,2-dichloroethane, tetrahydrofuran or ethyl acetate medium bases such as triethylamine, diisopropylethylamine, N-methylmorpholine or pyridine. It can be achieved by the reaction of acid XXI amine VIII in. Specific conditions are the use of propylphosphone anhydride and N-methylmorpholine in ethyl acetate for 18 hours at room temperature.

Step H: Removal of Boc N-protecting groups involves mineral acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or solvents at 0 ° C to 80 ° C, such as dichloromethane, chloroform, tetrahydrofuran, methanol, ethanol or organic acids in water, such as tri. This can be done with fluoroacetic acid, dichloroacetic acid, acetic acid or p-toluenesulfonic acid. A specific condition is the use of HCl in a mixture of dioxane and ethyl acetate for 18 hours at room temperature.

工程Ａ：アミンＶＩＩとエチレンジアミンＸＸＩＶとのアミドカップリングは、アミドカップリング試薬、例えばジシクロヘキシルカルボジイミド（ＤＣＣ）、１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）、Ｏ−（ベンゾトリアゾール−１−イル）−Ｎ，Ｎ，Ｎ’，Ｎ’−テトラメチルウロニウム−ヘキサフルオロホスフェート（ＨＢＴＵ），（１−［ビス（ジメチルアミノ）メチレン］−１Ｈ−１，２，３−トリアゾロ［４，５−ｂ］ピリジニウム ３−オキシド ヘキサフルオロホスフェート）（ＨＡＴＵ）、Ｏ−（ベンゾトリアゾール−１−イル）−Ｎ，Ｎ，Ｎ’，Ｎ’−テトラメチルウロニウム−テトラフルオロボレート（ＴＢＴＵ）、プロピルホスホン酸無水物（Ｔ３Ｐ）等、及び、溶媒、例えばメチレンクロリド、１，２−ジクロロエタン、テトラヒドロフラン、ジメチルホルムアミド又は酢酸エチル中塩基、例えばトリエチルアミン、ジイソプロピルエチルアミン、Ｎ−メチルモルホリン又はピリジンの存在下で達成することができる。特定の条件は、ＨＡＴＵと、ジメチルホルムアミド中ジイソプロピルエチルアミンを室温で１８時間使用することである。

Step A: The amide coupling of amine VII with ethylenediamine XXIV is performed by amide coupling reagents such as dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), O- (benzotriazole). -1-yl) -N, N, N', N'-tetramethyluronium-hexafluorophosphate (HBTU), (1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [ 4,5-b] Pyridinium 3-oxide hexafluorophosphate) (HATU), O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium-tetrafluoroborate (TBTU) In the presence of propylphosphonic acid anhydride (T3P) and the like, and solvents such as methylene chloride, 1,2-dichloroethane, tetrahydrofuran, dimethylformamide or ethyl acetate medium bases such as triethylamine, diisopropylethylamine, N-methylmorpholine or pyridine. Can be achieved with. Specific conditions are the use of HATU and diisopropylethylamine in dimethylformamide at room temperature for 18 hours.

Step B: Removal of Boc N-protecting groups involves mineral acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or solvents at 0 ° C to 80 ° C, such as dichloromethane, chloroform, tetrahydrofuran, methanol, ethanol or organic acids in water, such as tri. This can be done with fluoroacetic acid, dichloroacetic acid, acetic acid or p-toluenesulfonic acid. A specific condition is the use of HCl in a mixture of dioxane and ethyl acetate for 18 hours at room temperature.

工程Ａ：エステルＶＩは、適切な溶媒、例えば１，２−ジメトキシエタン、テトラヒドロフラン、トルエン、メタノール又はエタノール中還元剤、例えば水素化ホウ素リチウム、水素化ホウ素ナトリウム又はＲｅｄ−Ａｌでの−７８℃から還流で１〜２４時間の反応により、アルコールＸＸＶＩＩへ変換することができる。特定の条件は、メタノール中０℃で５時間の水素化ホウ素ナトリウムでの反応において使用され、混合物が一晩室温に温められることを可能にする。

Step A: The ester VI is from −78 ° C. in a suitable solvent such as 1,2-dimethoxyethane, tetrahydrofuran, toluene, methanol or a reducing agent in ethanol such as lithium borohydride, sodium borohydride or Red-Al. It can be converted to alcohol XXVII by reaction in reflux for 1 to 24 hours. Certain conditions are used in the reaction with sodium borohydride at 0 ° C. in methanol for 5 hours, allowing the mixture to warm to room temperature overnight.

Step B: Removal of Boc N-protecting groups involves mineral acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or solvents at 0 ° C to 80 ° C, such as dichloromethane, chloroform, tetrahydrofuran, methanol, ethanol or organic acids in water, such as tri. This can be done with fluoroacetic acid, dichloroacetic acid, acetic acid or p-toluenesulfonic acid. A specific condition is the use of HCl in a mixture of dioxane and ethyl acetate for 18 hours at room temperature.

Ｂｏｃ Ｎ−保護基の除去は、鉱酸、例えば塩酸、硫酸若しくはリン酸、又は、０℃から８０℃の溶媒、例えばジクロロメタン、クロロホルム、テトラヒドロフラン、メタノール、エタノール若しくは水中有機酸、例えばトリフルオロ酢酸、ジクロロ酢酸、酢酸若しくはｐ−トルエンスルホン酸によって行うことができる。特定の条件は、ジオキサンと酢酸エチルの混合物中ＨＣｌを室温で１８時間使用することである。

Boc N-protective groups can be removed with mineral acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or solvents at 0 ° C to 80 ° C, such as dichloromethane, chloroform, tetrahydrofuran, methanol, ethanol or organic acids in water, such as trifluoroacetic acid. This can be done with dichloroacetic acid, acetic acid or p-toluenesulfonic acid. A specific condition is the use of HCl in a mixture of dioxane and ethyl acetate for 18 hours at room temperature.

工程Ａ：アミンＶＩＩとアミンＸＸＸとのアミドカップリングは、アミドカップリング試薬、例えばジシクロヘキシルカルボジイミド（ＤＣＣ）、１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）、Ｏ−（ベンゾトリアゾール−１−イル）−Ｎ，Ｎ，Ｎ’，Ｎ’−テトラメチルウロニウム−ヘキサフルオロホスフェート（ＨＢＴＵ），（１−［ビス（ジメチルアミノ）メチレン］−１Ｈ−１，２，３−トリアゾロ［４，５−ｂ］ピリジニウム ３−オキシド ヘキサフルオロホスフェート）（ＨＡＴＵ）、Ｏ−（ベンゾトリアゾール−１−イル）−Ｎ，Ｎ，Ｎ’，Ｎ’−テトラメチルウロニウム−テトラフルオロボレート（ＴＢＴＵ）、プロピルホスホン酸無水物（Ｔ３Ｐ）等、及び、溶媒、例えばメチレンクロリド、１，２−ジクロロエタン、テトラヒドロフラン、ジメチルホルムアミド又は酢酸エチル中塩基、例えばトリエチルアミン、ジイソプロピルエチルアミン、Ｎ−メチルモルホリン又はピリジンの存在下で達成することができる。特定の条件は、ＨＡＴＵと、ジメチルホルムアミド中ジイソプロピルエチルアミンを室温で１８時間使用することである。

Step A: The amide coupling of amine VII and amine XXX is performed by amide coupling reagents such as dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), O- (benzotriazole). -1-yl) -N, N, N', N'-tetramethyluronium-hexafluorophosphate (HBTU), (1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [ 4,5-b] Pyridinium 3-oxide hexafluorophosphate) (HATU), O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium-tetrafluoroborate (TBTU) In the presence of propylphosphonic acid anhydride (T3P) and the like, and solvents such as methylene chloride, 1,2-dichloroethane, tetrahydrofuran, dimethylformamide or ethyl acetate medium bases such as triethylamine, diisopropylethylamine, N-methylmorpholine or pyridine. Can be achieved with. Specific conditions are the use of HATU and diisopropylethylamine in dimethylformamide at room temperature for 18 hours.

Step B: Removal of the Cbz group can be achieved by hydrogenation with a catalyst such as Pd-C in a solvent such as methanol or ethanol under standard or rising pressure. A specific condition is the use of a compound dissolved in hydrogen at atmospheric pressure, palladium on carbon as a catalyst and methanol at room temperature for 6 hours.

Step C: Removal of Boc N-protecting groups involves mineral acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or solvents at 0 ° C to 80 ° C, such as dichloromethane, chloroform, tetrahydrofuran, methanol, ethanol or organic acids in water, such as tri. This can be done with fluoroacetic acid, dichloroacetic acid, acetic acid or p-toluenesulfonic acid. A specific condition is the use of HCl in a mixture of dioxane and ethyl acetate for 18 hours at room temperature.

The method of carrying out the above reactions and processes will be apparent to those skilled in the art based on the present disclosure, or can be similarly estimated from the examples. The starting material is commercially available or can be made by a method similar to that described in the Examples below.

Crystalline When the compounds of the present invention are solid, those skilled in the art can have such compounds and salts thereof present in different crystalline or polymorphic forms, all of which are of the formulas specified in the present invention. You will understand that it falls within the scope.

Assay XIAP BIR2 and BIR3 TR-FRET Assay The ability of the test compound to inhibit the binding of the XIAP protein to peptide A (SMAC-derived peptide, described below) of the BIR2 and / or BIR3 domain is that the test compound is a SMAC-mimetic agent. As a result, it proves that it reactivates the apoptotic pathway of cells.

As described above (International Publication No. 2014/090709), the peptide AVPIAQKSEK (SEQ ID NO: 21) was considered optimal for use in this assay. The peptide sequence was derivatized with biotin to give AVPIAQKSEK- (ε-biotin) -OH 1: 2 TFA as a substrate for the TR-FRET assay.

The XIAP protein sequence was obtained from the SWISS-PROT protein sequence database, from which the BIR2 and BIR3 domains were derived. The sequence of the BIR2 domain used in the TR-FRET assay
MRHHHHHRDHFALDRPSETHADYLLRTGQVVDISDTIYPRNPAMYSEEARLKSFQNWPDYAHLTPELLASAGLYYTGIGDQVQCFACGGKGKLKNWEPGDRAWSWSEHRRHRFPNCFFVLGN

The sequence of the BIR3 domain used in the TR-FRET assay
MRHHHHHHRSDAVSSDRNFPNSTNLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGFYALGEGDDKVKCFHCGGGLTDWWKPSEDPWEQHAKWYPGCKYLLEEQKGQEYINNIHLTHSLEF.

For the Bir2 assay, the BIR2 domain tagged with 3.5 nanomoles of 6x histidine, corresponding to amino acids 124-240 of XIAP, was subjected to 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol. In the presence of (DTT) and 0.1 mg / mL bovine serum albumin (BSA), it was mixed with 3.6 nM AVPIAQKSEK- (ε-biotin) -OH 1: 2 TFA. Following an incubation at 37 ° C. for 45 minutes, the final concentrations of europium-streptavidin (Eu-8044 streptavidin, PerkinElmer) and allophycocyanin (Columbia Biosciences) -conjugated anti-histidine antibodies reached 0.6 nM and 1 nM, respectively. It was added until it became. The time-resolved fluorescence resonance energy transfer (TR-FRET) signal was measured at room temperature after 1 hour. The efficacy of the test compound was assessed at 10 consecutively diluted concentrations. Determine the percent inhibition at each concentration to produce an IC ₅₀ value of each test compound.

For the Bir3 assay, 13.3 nanomoles of BIR3 domain, corresponding to amino acids 241-356 of XIAP, 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg. In the presence of / mL bovine serum albumin (BSA), it was mixed with 25.2 nM AVPIAQKSEK- (ε-biotin) -OH 1: 2 TFA. Following incubation at 37 ° C. for 45 minutes, europium-streptavidin and allophycocyanin-conjugated anti-histidine antibodies were added until final concentrations were 0.6 nM and 2.5 nM, respectively. The time-resolved fluorescence resonance energy transfer (TR-FRET) signal was measured at room temperature after 0.5 hours. The efficacy of the test compound was assessed at 10 consecutively diluted concentrations. Determine the percent inhibition at each concentration to produce an IC ₅₀ value of each test compound.

TR-FRET assay for cIAP BIR2 and BIR3 The peptide AVPIAQKSEK was derivatized with biotin to give AVPIAQKSEK- (ε-biotin) -OH 1: 2 TFA as a substrate for the TR-FRET assay.

The cIAP protein sequence was obtained from the SWISS-PROT protein sequence database, from which the BIR2 and BIR3 domains were derived. The sequence of the BIR2 domain used in the TR-FRET assay
MGSSHHHHHHSSGLVPRGSHMASENLYFQGTNPYSYAMSTEEARFLTYHMWPLTFLSPSELARAGFYYIGPGDRVACFAC GGKLSNWEDK DDAMSEHRRHFPNCPFLENS LET.

The sequence of the BIR3 domain used in the TR-FRET assay
MAHHHHHHENLYFQGS S ISNLSMQTHAARMRTFMYWPSSVPVQPEQLASAGFYYVGRNDDVKCFCDGGLRCWESGDDPWVEHAKWFPRCEFLIRMKGQEFVDEIQGRY.

For the Bir2 assay, the BIR2 domain tagged with 12 nanomoles of 6xhistidine, corresponding to amino acids 174-256 of cIAP, was subjected to 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol (DTT). ) And 0.1 mg / mL bovine serum albumin (BSA), mixed with 19.3 nM AVPIAQKSEK- (ε-biotin) -OH 1: 2 TFA. Following incubation at 37 ° C. for 45 minutes, europium-streptavidin and allophycocyanin-conjugated anti-histidine antibodies were added until final concentrations of 0.6 nM and 4 nM, respectively, and incubated at room temperature for 60 minutes. After a final overnight incubation at 4 ° C., time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured. The titer of the test compound was assessed at 10 consecutively diluted concentrations. Determine the percent inhibition at each concentration to produce an IC ₅₀ value of each test compound.

For the Bir3 assay, 25.5 nanomoles of BIR3 domain corresponding to amino acid 260-352 of cIAP, 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg. In the presence of / mL bovine serum albumin (BSA), it was mixed with 41 nM AVPIAQKSEK- (ε-biotin) -OH 1: 2 TFA. Following incubation at 37 ° C. for 45 minutes, europium-streptavidin and allophycocyanin-conjugated anti-histidine antibodies were added to final concentrations of 0.6 nM and 6.4 nM, respectively, and incubated at room temperature for 60 minutes. After a final overnight incubation at 4 ° C., time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured. The titer of the test compound was assessed at 10 consecutively diluted concentrations. Determine the percent inhibition at each concentration to produce an IC ₅₀ value of each test compound.

Cell Viability Assay SK-OV-3 cells and HCT-116 cells were obtained from American Type Culture Collection (ATCC, 10801 University Boulevard Manassas, VA 20110 USA) and supplemented with 10% FBS and 2 mmol / L L-glutamine. The McCoy was maintained in a 5A medium. All media and supplements were purchased from Invitrogen (Life Technologies GmbH, Frankfurter Strase 129B 64293 Darmstadt Germany).

By adding 3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) according to the manufacturer's protocol (Sigma-Aldrich Chemie GmbH, Eschenstr. 5 82024 Taufkirchen Germany). , Cell viability was evaluated. For single drug activity, SK-OV-3 cells were seeded in 96-well plates (BD Biosciences, Tullastrasse 8-1269126 Heidelberg Germany) at 2000 cells per well, and after 24 hours, indicated concentrations of N- (2). -(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-) 2H-pyran-4-yl) acetyl) isoindoline-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)-)- Incubated with 5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indoline-3-carboxamide trihydrochloride (Example 2). The compounds were stored in aliquots at −20 ° C. as a stock solution of 10 mM in DMSO. For the combination, HCT-116 cells were seeded in 96-well plates (BD Biosciences, Tullastrasse 8-12 69126 Heidelberg Germany) with 2000 cells per well, and after 24 hours, the indicated concentration of N- (2-((((((() S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-) 4-yl) acetyl) isoindoline-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-methyl-) 2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indoline-3-carboxamide trihydrochloride alone or DR5-FAP bispecific antibody X AB (DR5 binder: Incubated in combination with VH SEQ ID NO: 7, VL SEQ ID NO: 8, FAP binding agent: VH SEQ ID NO: 15, VL SEQ ID NO: 16). After 5 days of incubation, cells were treated with MTT for 1 hour and the dye was extracted with ethanol. Absorbance was measured at 570 nm (Tecan Infinite 200 PRO, Tecan Group Ltd. Seestrase 103 8708 Mannedorf Switzerland) and a reference wavelength of 620 nm was used. All assays were duplicated, data were standardized and controlled, and analyzed using XLfit software.

Determine the percent inhibition at each concentration to produce an IC ₅₀ value of each test compound. These values are listed in the table below.

Brief Description of Drawings Figure 1: N-(2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) in cell viability assay for SK-OV-3 cells ) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-5-yl) amino) -2-oxoethyl) -6 -(4-Fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) ) Single drug activity of indoline-3-carboxamide trihydrochloride.

FIG. 2: N- (2-((((() S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-) 4-yl) acetyl) isoindoline-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-methyl-) 2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) Indoline-3-carboxamide The activity of trihydrochloride.

FIG. 3: As a combination of a single agent and the DR5-FAP bispecific antibody XAB (fixed concentration of FAP-DR5, 1.5 μg / ml) in the cell viability assay of HCT-116 cells). , N-(2-(((S) -3-((2,6-difluorophenyl) carboxamide) -2-((S) -2-((S) -2- (methylamino) propanamide))- 2- (Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1-(2-(((((4-fluorobenzyl) -3-methyl-1-yl) 2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) Indoline-3-carboxamide Trihydrochloride activity.

FIG. 4: Schematic diagram of FAP-DR5 bispecific antibody molecular design and mechanism of action.

The compounds of the present invention can be synthesized according to known techniques. The following examples and references are provided to aid in the understanding of the present invention. However, the examples are not intended to limit the invention and the true scope of the invention is described in the appended claims.

Ｂｏｃ無水物（１．４９ｇ、１．５９ｍｌ、６．８４ｍｍｏｌ、当量：１．０９）をジクロロメタン（３０ｍｌ）中メチル ６−ブロモ−３−メチルインドリン−３−カルボキシレート（１．６９６ｇ、６．２８ｍｍｏｌ、当量：１）（国際公開第２０１２１４３７２６号に記載）とＤＩＰＥＡ（９３３ｍｇ、１．２６ｍｌ、７．２２ｍｍｏｌ、当量：１．１５）の溶液に０℃で添加した。室温にゆっくりと温めながら、週末にかけて撹拌を続けた。飽和ＮａＨＣＯ_３溶液を添加し、ジクロロメタンで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤としてヘプタン／酢酸エチル（０−５０％酢酸エチル）を使用したカラムクロマトグラフィーにより精製し、１−ｔｅｒｔ−ブチル ３−メチル ６−ブロモ−３−メチル−インドリン−１，３−ジカルボキシレート（１．７２５ｇ、７４．２％）を明黄色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝３５７．０ Example 1
N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-) 2H-pyran-4-yl) acetyl) isoindolin-5-yl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-methyl-2- ( ((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride a) 1-tert-butyl 3-methyl 6-bromo-3-methyl-indolin-1, 3-Dicarboxylate

Boc anhydride (1.49 g, 1.59 ml, 6.84 mmol, equivalent: 1.09) in dichloromethane (30 ml) methyl 6-bromo-3-methylindoline-3-carboxylate (1.696 g, 6.28 mmol) Equivalent: 1) (described in WO 2012143726) and DIPEA (933 mg, 1.26 ml, 7.22 mmol, equivalent: 1.15) were added at 0 ° C. Stirring continued over the weekend, slowly warming to room temperature. Saturated NaHCO ₃ solution was added, extracted 3 times with dichloromethane, dried over magnesium sulphate, filtered and evaporated. The crude material was applied to silica gel, purified by column chromatography using heptane / ethyl acetate (0-50% ethyl acetate) as eluent, and 1-tert-butyl 3-methyl 6-bromo-3-methyl-indrin. -1,3-dicarboxylate (1.725 g, 74.2%) was obtained as a bright yellow oil. MS: m / z (M + H) ⁺ = 357.0

（ｄｐｐｆ）ＰｄＣｌ_２．ＣＨ_２Ｃｌ_２（５６ｍｇ、６８．６μｍｏｌ、当量：０．０１）とＴＨＦ（２７．４ｍｌ、１３．７ｍｍｏｌ、当量：２）中（４−フルオロベンジル）塩化亜鉛（ＩＩ）の溶液とを、乾燥ＴＨＦ（１３．９ｍｌ）中１−ｔｅｒｔ−ブチル ３−メチル ６−ブロモ−３−メチルインドリン−１，３−ジカルボキシレート（２．５３９ｇ、６．８６ｍｍｏｌ、当量：１）の溶液に添加した。混合物を密閉バイアル中９０℃で一晩撹拌した。水を数滴添加した後、粗混合物をシリカゲルに適用し、溶出剤としてヘプタン／酢酸エチル（０−３０％酢酸エチル）を使用したカラムクロマトグラフィーにより精製し、１−ｔｅｒｔ−ブチル ３−メチル ６−（４−フルオロベンジル）−３−メチルインドリン−１，３−ジカルボキシレート（１．４４ｇ、５２．６％）を明黄色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝３９９．１ b) tert-Butyl-methyl 6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1,3-dicarboxylate

(Dppf) PdCl ₂ . A solution of _{CH 2 Cl 2} (56 mg, 68.6 μmol, equivalent: 0.01) and THF (27.4 ml, 13.7 mmol, equivalent: 2) in (4-fluorobenzyl) zinc chloride (II) was dried. It was added to a solution of 1-tert-butyl 3-methyl 6-bromo-3-methylindrin-1,3-dicarboxylate (2.539 g, 6.86 mmol, equivalent: 1) in THF (13.9 ml). The mixture was stirred in a closed vial at 90 ° C. overnight. After adding a few drops of water, the crude mixture was applied to silica gel and purified by column chromatography using heptane / ethyl acetate (0-30% ethyl acetate) as eluent, 1-tert-butyl 3-methyl 6 -(4-Fluorobenzyl) -3-methylindrin-1,3-dicarboxylate (1.44 g, 52.6%) was obtained as a bright yellow oil. MS: m / z (M + H) ⁺ = 399.1

ジオキサン（８．６３ｇ、８．２１ｍｌ、３２．９ｍｍｏｌ、当量：９．１２）中塩酸を、メタノール（１０ｍｌ）中１−ｔｅｒｔ−ブチル ３−メチル ６−（４−フルオロベンジル）−３−メチルインドリン−１，３−ジカルボキシレート（１．４４ｇ、３．６ｍｍｏｌ、当量：１）に添加し、５０℃で５時間撹拌した。反応混合物を真空中で濃縮し、残留物を水と酢酸エチルとに分けた。水性相を飽和水性ＮａＨＣＯ_３でクエンチし、固体を濾別した。水性相を酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤としてヘプタン／酢酸エチル（０−５０％酢酸エチル）を使用したカラムクロマトグラフィーにより精製し、メチル ６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボキシレート（１．０５ｇ、９７．３％）を明褐色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝３００．１４ c) Methyl 6-[(4-fluorophenyl) methyl] -3-methyl-indoline-3-carboxylate

Hydrochloric acid in dioxane (8.63 g, 8.21 ml, 32.9 mmol, equivalent: 9.12), 1-tert-butyl 3-methyl 6- (4-fluorobenzyl) -3-methyl indoline in methanol (10 ml). It was added to -1,3-dicarboxylate (1.44 g, 3.6 mmol, equivalent: 1) and stirred at 50 ° C. for 5 hours. The reaction mixture was concentrated in vacuo and the residue was separated into water and ethyl acetate. The aqueous phase was _{quenched with saturated aqueous NaHCO 3} and the solids were filtered off. The aqueous phase was extracted 3 times with ethyl acetate, dried over magnesium sulphate, filtered and evaporated. The crude material was applied to silica gel and purified by column chromatography using heptane / ethyl acetate (0-50% ethyl acetate) as an eluent, and methyl 6-[(4-fluorophenyl) methyl] -3-methyl- Indolin-3-carboxylate (1.05 g, 97.3%) was obtained as a light brown oil. MS: m / z (M + H) ⁺ = 30.14

メチル ６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキシレート（４０３ｍｇ、１．３５ｍｍｏｌ、当量：１）を酢酸エチル（４．５ｍｌ）に溶解し、２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）酢酸（５００ｍｇ、１．３５ｍｍｏｌ、当量：１）（国際公開第２０１２１４３７２６号に記載）、Ｎ−メチルモルホリン（１．３６ｇ、１．４８ｍｌ、１３．５ｍｍｏｌ、当量：１０）及び ＥｔＯＡｃ ５０％（２．５７ｇ、２．４ｍｌ、４．０４ｍｍｏｌ、当量：３）中Ｔ３Ｐを添加し、室温で一晩撹拌した。反応混合物を酢酸エチルで希釈し、飽和ＮａＨＣＯ_３で洗浄し、Ｎａ_２ＳＯ４で乾燥させ、真空中で濃縮した。粗物質をシリカゲルに適用し、溶出剤としてヘプタン／酢酸エチル（０−５０％酢酸エチル）を使用したカラムクロマトグラフィーにより精製し、メチル １−［２−［（２Ｓ，５Ｒ）−４−ｔｅｒｔ−ブトキシカルボニル−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボキシレート（５８８ｍｇ、６６．９％）を白色の泡として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６５３．３７ d) Methyl 1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazine-1-yl] ] Acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carboxylate

Methyl 6- (4-fluorobenzyl) -3-methylindrin-3-carboxylate (403 mg, 1.35 mmol, equivalent: 1) was dissolved in ethyl acetate (4.5 ml) and 2-((2S, 5R)). -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetate (500 mg, 1.35 mmol, equivalent: 1) (International release) In No. 2012143726), N-methylmorpholine (1.36 g, 1.48 ml, 13.5 mmol, equivalent: 10) and 50% EtOAc (2.57 g, 2.4 ml, 4.04 mmol, equivalent: 3). T3P was added and stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate _{, washed with saturated NaHCO 3} , dried over Na ₂ SO4 and concentrated in vacuo. The crude material was applied to silica gel and purified by column chromatography using heptane / ethyl acetate (0-50% ethyl acetate) as eluent and methyl 1- [2-[(2S, 5R) -4-tert-. Butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl -Indoline-3-carboxylate (588 mg, 66.9%) was obtained as white foam. MS: m / z (M + H) ⁺ = 653.37

メチル １−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキシレート（２．１ｇ、３．２２ｍｍｏｌ、当量：１）をエタノール（１３ｍｌ）及び水（５．２ｍｌ）中で撹拌した。水酸化リチウム（２３１ｍｇ、９．６５ｍｍｏｌ、当量：３）を添加し、６０℃で３時間撹拌を続けた。溶媒をエバポレートし、ＨＣｌ １Ｎ ａｑを用いて水相をｐＨ７に酸性化した。混合物を酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートして、１−［２−［（２Ｓ，５Ｒ）−４−ｔｅｒｔ−ブトキシカルボニル−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボン酸（２．０４ｇ、９９．３％）をオフホワイトの泡として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６３９．３５ e) 1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazine-1-yl] Acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carboxylic acid

Methyl 1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazine-1-yl) acetyl)- 6- (4-Fluorobenzyl) -3-methylindoline-3-carboxylate (2.1 g, 3.22 mmol, equivalent: 1) was stirred in ethanol (13 ml) and water (5.2 ml). Lithium hydroxide (231 mg, 9.65 mmol, equivalent: 3) was added and stirring was continued at 60 ° C. for 3 hours. The solvent was evaporated and the aqueous phase was acidified to pH 7 with HCl 1N aq. The mixture was extracted 3 times with ethyl acetate, dried over magnesium sulfate, filtered, evaporated and 1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[ (3R) -3-methylmorpholin-4-yl] methyl] piperazine-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indrin-3-carboxylic acid (2.04 g) , 99.3%) was obtained as an off-white foam. MS: m / z (M + H) ⁺ = 639.35

１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（１００ｍｇ、１５７μｍｏｌ、当量：１）を、酢酸エチル（６００μｌ）中でｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（９６．４ｍｇ、１５７μｍｏｌ、当量：１；中間体２）、Ｎ−メチルモルホリン（１５８ｍｇ、１７２μｌ、１．５７ｍｍｏｌ、当量：１０）及びＥｔＯＡｃ ５０％（２９９ｍｇ、２８０μｌ、４７０μｍｏｌ、当量：３）中Ｔ３Ｐと室温で一晩撹拌した。飽和ＮａＨＣＯ_３溶液を添加し、酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチルを使用したカラムクロマトグラフィーにより精製し、３−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（１２０ｍｇ、６２％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２３６．２３ f) 3-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl -Acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindoline-5-yl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl]- 2-Oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate

1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6 -(4-Fluorobenzyl) -3-methylindrin-3-carboxylic acid (100 mg, 157 μmol, equivalent: 1) was added to tert-butyl ((S) -1-(((S)) in ethyl acetate (600 μl). -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindrin-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) Ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (96.4 mg, 157 μmol, equivalent: 1; intermediate 2), N-methylmorpholin (158 mg, 172 μl, 1.57 mmol, equivalent: 10) ) And EtOAc in 50% (299 mg, 280 μl, 470 μmol, equivalent: 3) with T3P and stirred overnight at room temperature. Saturated NaHCO ₃ solution was added, extracted 3 times with ethyl acetate, dried over magnesium sulfate, filtered and evaporated. The crude substance was applied to silica gel and purified by column chromatography using ethyl acetate as an eluent, and 3-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert -Butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindoline-5-yl] carbamoyl] -6- [(4-Fluorophenyl) methyl] -3-methyl-isoindoline-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl ] Piperazin-1-carboxylate (120 mg, 62%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1236.23

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（１１０ｍｇ、８９μｍｏｌ、当量：１）を酢酸エチル（１．５ｍｌ）中でジオキサン４Ｍ（２ｍｌ、８ｍｍｏｌ、当量：８９．９）中ＨＣｌと室温で一晩撹拌した。沈殿物を濾別し、エーテルで洗浄した。高減圧下で固体を乾燥させ、Ｎ−［（３Ｓ）−３−［（２，６−ジフルオロフェニル）カルバモイル］−２−［（２Ｓ）−２−［［（２Ｓ）−２−（メチルアミノ）プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］イソインドリン−５−イル］−６−［（４−フルオロフェニル）メチル］−３−メチル−１−［２−［（２Ｒ，５Ｒ）−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］インドリン−３−カルボキサミド トリヒドロクロリド（９９．４ｍｇ、９７．５％）をオフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０３６．５２ g) N-[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino) propanoyl] amino] -2] -Tetrahydropyran-4-yl-acetyl] isoindoline-5-yl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl- 2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indoline-3-carboxamide trihydrochloride

(2R, 5S) -tert-butyl 4-(2-(3-(((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) ) Propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) -3-((2,6-difluorophenyl) carbamoyl) isoindolin-5-yl) carbamoyl) -6- (4-fluoro) Benzyl) -3-methylindolin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (110 mg, 89 μmol, equivalent:) 1) was stirred in ethyl acetate (1.5 ml) with HCl in Dioxane 4M (2 ml, 8 mmol, equivalent: 89.9) overnight at room temperature. The precipitate was filtered off and washed with ether. The solid was dried under high pressure and N-[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino)). ) Propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindoline-5-yl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 2R,) 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indoline-3-carboxamide trihydrochloride (99.4 mg, 97.5) %) Was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 1036.52

１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（５００ｍｇ、７８３μｍｏｌ、当量：１）を、メチル ２−アミノアセテート ヒドロクロリド（１１３ｍｇ）、ＤＩＰＥＡ（４０５ｍｇ、５４７μｌ、９００μｍｏｌ、当量：１．１５）及びＨＡＴＵ（３８７ｍｇ、１．０２ｍｍｏｌ、当量：１．３）と室温で一晩撹拌した。反応混合物を０．５Ｍ ＨＣｌ ａｑ．に注ぎ、酢酸エチルで３回抽出し、ブラインで洗浄し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−５％メタノール）を使用したカラムクロマトグラフィーにより精製し、（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（６−（４−フルオロベンジル）−３−（（２−メトキシ−２−オキソエチル）カルバモイル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（３６９ｍｇ、５２０μｍｏｌ、６６．４％）を明黄色の泡として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝７１０．３９ Example 2
N-(2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R) , 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride a) (2R, 5S) -tert-butyl 4- (2- (6- (4-Fluorobenzyl) -3-((2-methoxy-2-oxoethyl) carbamoyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5 -(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate

1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6 -(4-Fluorobenzyl) -3-methylindrin-3-carboxylic acid (500 mg, 783 μmol, equivalent: 1), methyl 2-aminoacetate hydrochloride (113 mg), DIPEA (405 mg, 547 μl, 900 μmol, equivalent: 1) .15) and HATU (387 mg, 1.02 mmol, equivalent: 1.3) were stirred overnight at room temperature. The reaction mixture was added to 0.5 M HCl aq. Pour into, extracted 3 times with ethyl acetate, washed with brine, dried over magnesium sulphate, filtered and evaporated. The crude material was applied to silica gel and purified by column chromatography using ethyl acetate / methanol (0-5% methanol) as eluent, and (2R, 5S) -tert-butyl 4- (2- (6-(6-). 4-Fluorobenzyl) -3-((2-Methoxy-2-oxoethyl) carbamoyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-) Methylmorpholino) methyl) piperazin-1-carboxylate (369 mg, 520 μmol, 66.4%) was obtained as a bright yellow foam. MS: m / z (M + H) ⁺ = 710.39

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（６−（４−フルオロベンジル）−３−（（２−メトキシ−２−オキソエチル）カルバモイル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（３６０ｍｇ、５０７μｍｏｌ、当量：１）を、エタノール（２．５ｍｌ）及び水（１ｍｌ）中で撹拌した。水酸化リチウム（３６．４ｍｇ、１．５２ｍｍｏｌ、当量：３）を添加し、６０℃で３時間撹拌を続けた。溶媒をエバポレートし、ＨＣｌ ２Ｎ ａｑを用いて水相をｐＨ５〜６に酸性化した。混合物を酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートして、２−（１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（３０６ｍｇ、４４０μｍｏｌ、８６．７％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６９６．３８ b) (2- (1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazine-1-) Il) Acetyl) -6- (4-Fluorobenzyl) -3-methylindoline-3-carboxamide) acetic acid

(2R, 5S) -tert-butyl 4-(2- (6- (4-fluorobenzyl) -3-((2-methoxy-2-oxoethyl) carbamoyl) -3-methylindolin-1-yl) -2 -Oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazine-1-carboxylate (360 mg, 507 μmol, equivalent: 1), ethanol (2.5 ml) and water (1 ml). ) Stirred in. Lithium hydroxide (36.4 mg, 1.52 mmol, equivalent: 3) was added and stirring was continued at 60 ° C. for 3 hours. The solvent was evaporated and the aqueous phase was acidified to pH 5-6 with HCl 2N aq. The mixture was extracted 3 times with ethyl acetate, dried over magnesium sulfate, filtered and evaporated to 2-(1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl). -2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxamide) acetic acid (306 mg, 440 μmol, 86) .7%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 696.38

２−（１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（５０ｍｇ、７１．９μｍｏｌ、当量：１）を、酢酸エチル（３００μｌ）中でｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（４４．２ｍｇ、７１．９μｍｏｌ、当量：１；中間体２）、Ｎ−メチルモルホリン（７２．７ｍｇ、７９μｌ、７１９μｍｏｌ、当量：１０）及びＥｔＯＡｃ ５０％（１３７ｍｇ、１２８μｌ、２１６μｍｏｌ、当量：３）中Ｔ３Ｐと室温で一晩撹拌した。飽和ＮａＨＣＯ_３溶液を添加し、酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−１０％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［３−［［２−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］アミノ］−２−オキソ−エチル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（４８ｍｇ、５１．６％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）＋＝１２９３．６５ c) tert-butyl (2R, 5S) -4- [2- [3-[[2-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxy) Carbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino] -2-oxo- Ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methyl Morphorin-4-yl] Methyl] Piperazin-1-carboxylate

2-(1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) ) -6- (4-Fluorobenzyl) -3-methylindrin-3-carboxamide) acetic acid (50 mg, 71.9 μmol, equivalent: 1) in ethyl acetate (300 μl) with tert-butyl ((S) -1). -(((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindolin-2-yl) -2-oxo-1- (tetrahydro-2H-) Piran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (44.2 mg, 71.9 μmol, equivalent: 1; intermediate 2), N-methylmorpholin (72.7 mg) , 79 μl, 719 μmol, equivalent: 10) and EtOAc in 50% (137 mg, 128 μl, 216 μmol, equivalent: 3) with T3P and stirred overnight at room temperature. Saturated NaHCO ₃ solution was added, extracted 3 times with ethyl acetate, dried over magnesium sulfate, filtered and evaporated. The crude material was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-10% methanol) as eluent, and tert-butyl (2R, 5S) -4- [2- [3- [ [2-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl- Acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindolin-5-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl -Indoline-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (48 mg, 51.6) %) Was obtained as a white solid. MS: m / z (M + H) + = 1293.65

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレートを、酢酸エチル（６００μＬ）中でジオキサン４Ｍ（７５０μｌ、３ｍｍｏｌ、当量：９２．４）中ＨＣｌと室温で一晩撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄した。高減圧下で固体を乾燥させ、Ｎ−［２−［［（３Ｓ）−３−［（２，６−ジフルオロフェニル）カルバモイル］−２−［（２Ｓ）−２−［［（２Ｓ）−２−（メチルアミノ）プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］イソインドリン−５−イル］アミノ］−２−オキソ−エチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−１−［２−［（２Ｒ，５Ｒ）−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］インドリン−３−カルボキサミド トリヒドロクロリド（３５．５ｍｇ、９０．９％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０９３．５４ d) N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino) propanoyl]] Amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [ 2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide trihydrochloride

(2R, 5S) -tert-butyl 4-(2-(3-((2-(((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl)) (Methyl) Amino) Propanamide) -2- (Tetrahydro-2H-Pyran-4-yl) Acetyl) -3-((2,6-Difluorophenyl) Carbamoyl) Isoindrin-5-yl) Amino) -2- Oxoethyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin- The 1-carboxylate was stirred overnight with HCl in dioxane 4M (750 μl, 3 mmol, equivalent: 92.4) in ethyl acetate (600 μL) at room temperature. The precipitate was filtered off and washed with diethyl ether. The solid was dried under high vacuum and N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2]. -(Methylamino) propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) methyl]- 3-Methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3 -Carboxamide trihydrochloride (35.5 mg, 90.9%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1093.54

１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（７５ｍｇ、１１７μｍｏｌ、当量：２）を、ＤＭＦ（５００μｌ）中でエタン−１，２−ジアミン（３．５３ｍｇ、３．９３μｌ、５８．７μｍｏｌ、当量：１）、ＤＩＰＥＡ（６０．７ｍｇ、７９．９μｌ、４７０μｍｏｌ、当量：８）及びＨＡＴＵ（５８．１ｍｇ、１５３μｍｏｌ、当量：２．６）と室温で一晩撹拌した。混合物を０．５Ｍ ＨＣｌ ａｑ．でクエンチし、酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−１０％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［３−［２−［［１−［２−［（２Ｓ，５Ｒ）−４−ｔｅｒｔ−ブトキシカルボニル−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボニル］アミノ］エチルカルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（５６ｍｇ、７３．２％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１３０１．７５ Example 3
a) (R, R, R) -N, N'-(ethane-1,2-diyl) bis (6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)) -5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) trihydrochloride tert-butyl (2R, 5S) -4- [2 -[3- [2-[[1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] ] Piperazin-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indrin-3-carbonyl] amino] ethylcarbamoyl] -6-[(4-fluorophenyl) methyl]- 3-Methyl-Indoline-1-yl] -2-oxo-Ethyl] -2-Methyl-5-[[(3R) -3-methylmorpholin-4-yl] Methyl] Piperazin-1-carboxylate

1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6 -(4-Fluorobenzyl) -3-methylindrin-3-carboxylic acid (75 mg, 117 μmol, equivalent: 2) in DMF (500 μl) with ethane-1,2-diamine (3.53 mg, 3.93 μl, It was stirred overnight at room temperature with 58.7 μmol, equivalent: 1), DIPEA (60.7 mg, 79.9 μl, 470 μmol, equivalent: 8) and HATU (58.1 mg, 153 μmol, equivalent: 2.6). Mixture 0.5M HCl aq. It was quenched with, extracted 3 times with ethyl acetate, dried over magnesium sulfate, filtered and evaporated. The crude material was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-10% methanol) as eluent, and tert-butyl (2R, 5S) -4- [2- [3- [ 2-[[1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-] Il] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carbonyl] amino] ethylcarbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indrin -1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (56 mg, 73.2%) Was obtained as a white solid. MS: m / z (M + H) ⁺ = 1301.75

（Ｒ，２Ｒ，２’Ｒ，５Ｓ，５’Ｓ）−ジ−ｔｅｒｔ−ブチル ４，４’−（（３，３’−（（エタン−１，２−ジイルビス（アザンジイル））ビス（カルボニル））ビス（６−（４−フルオロベンジル）−３−メチルインドリン−３，１−ジイル））ビス（２−オキソエタン−２，１−ジイル））ビス（２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート）（４９ｍｇ、３７．６μｍｏｌ、当量：１）を、酢酸エチル（７５０μｌ）中で、ジオキサン４Ｍ（８５０μｌ、３．４ｍｍｏｌ、当量：９０．３）中 ＨＣｌと室温で一晩撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄した。固体を高減圧下で乾燥させ、（Ｒ，Ｒ，Ｒ）−Ｎ，Ｎ’−（エタン−１，２−ジイル）ビス（６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド）トリヒドロクロリド（３９．４ｍｇ、８６．４％）をオフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１１０１．６５ b) 6-[(4-fluorophenyl) methyl] -N- [2-[[6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5) -Methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carbonyl] amino] ethyl] -3-methyl-1- [2-] [(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide trihydrochloride

(R, 2R, 2'R, 5S, 5'S) -Di-tert-butyl 4,4'-((3,3'-((ethane-1,2-diylbis (azandiyl)) bis (carbonyl)) ) Bis (6- (4-fluorobenzyl) -3-methylindrin-3,1-diyl)) Bis (2-oxoethane-2,1-diyl)) Bis (2-methyl-5-(((R)) -3-Methylmorpholino) Methyl) Piperazine-1-carboxylate) (49 mg, 37.6 μmol, equivalent: 1) in ethyl acetate (750 μl) with dioxane 4M (850 μl, 3.4 mmol, equivalent: 90.3). ) Medium HCl was stirred at room temperature overnight. The precipitate was filtered off and washed with diethyl ether. The solid was dried under high pressure and (R, R, R) -N, N'-(ethane-1,2-diyl) bis (6- (4-fluorobenzyl) -3-methyl-1- (2). -((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazine-1-yl) acetyl) indolin-3-carboxamide) trihydrochloride (39.4 mg, 86) .4%) was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 1101.65

１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（３００ｍｇ、４７０μｍｏｌ、当量：１）を、メチル ４−アミノベンゾエート（７１ｍｇ、４７０μｍｏｌ、当量：１．０）、ＤＩＰＥＡ（１８２ｍｇ、２４６μｌ、１．４１ｍｍｏｌ、当量：３）及びＨＡＴＵ（２３２ｍｇ、６１１μｍｏｌ、当量：１．３）と室温で一晩撹拌した。反応混合物を０．５Ｍ ＨＣｌ ａｑ．に注ぎ、酢酸エチルで３回抽出し、ブラインで洗浄し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤としてヘプタン／酢酸エチル（０−１００％酢酸エチル）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［６−［（４−フルオロフェニル）メチル］−３−［（４−メトキシカルボニルフェニル）カルバモイル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（１８２ｍｇ、５０．２％）をオレンジ色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝７７２．４ Example 4
N-(4-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-5-yl) carbamoyl) phenyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)) -5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride a) tert-butyl (2R, 5S) -4-[ 2- [6-[(4-fluorophenyl) methyl] -3-[(4-methoxycarbonylphenyl) carbamoyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl- 5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate

1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6 -(4-Fluorobenzyl) -3-methylindrin-3-carboxylic acid (300 mg, 470 μmol, equivalent: 1), methyl 4-aminobenzoate (71 mg, 470 μmol, equivalent: 1.0), DIPEA (182 mg, 246 μl) , 1.41 mmol, equivalent: 3) and HATU (232 mg, 611 μmol, equivalent: 1.3) and stirred overnight at room temperature. The reaction mixture was added to 0.5 M HCl aq. Pour into, extracted 3 times with ethyl acetate, washed with brine, dried over magnesium sulphate, filtered and evaporated. The crude material was applied to silica gel, purified by column chromatography using heptane / ethyl acetate (0-100% ethyl acetate) as eluent, and tert-butyl (2R, 5S) -4- [2- [6-]. [(4-Fluorophenyl) Methyl] -3-[(4-Methoxycarbonylphenyl) Carbamoyl] -3-Methyl-Indoline-1-yl] -2-oxo-Ethyl] -2-Methyl-5-[[( 3R) -3-methylmorpholin-4-yl] methyl] piperazine-1-carboxylate (182 mg, 50.2%) was obtained as an orange oil. MS: m / z (M + H) ⁺ = 772.4

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（６−（４−フルオロベンジル）−３−（（４−（メトキシカルボニル）フェニル）カルバモイル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（１７５ｍｇ、２２７μｍｏｌ、当量：１）をエタノール（１．２ｍｌ）及び水（５００μｌ）中で撹拌した。水酸化リチウム（１６．３ｍｇ、６８０μｍｏｌ、当量：３）を添加し、６０℃で３時間撹拌を続けた。溶媒をエバポレートし、ＨＣｌ １Ｎ ａｑを用いて水層をｐＨ６に酸性化した。混合物を酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートして、４−［［１−［２−［（２Ｓ，５Ｒ）−４−ｔｅｒｔ−ブトキシカルボニル−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボニル］アミノ］安息香酸（８７ｍｇ、５０．６％）をオフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝７５８．４ b) 4-[[1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazine- 1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carbonyl] amino] benzoic acid

(2R, 5S) -tert-butyl 4-(2- (6- (4-fluorobenzyl) -3-((4- (methoxycarbonyl) phenyl) carbamoyl) -3-methylindrin-1-yl) -2 -Oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazine-1-carboxylate (175 mg, 227 μmol, equivalent: 1) in ethanol (1.2 ml) and water (500 μl) Stirred in. Lithium hydroxide (16.3 mg, 680 μmol, equivalent: 3) was added and stirring was continued at 60 ° C. for 3 hours. The solvent was evaporated and the aqueous layer was acidified to pH 6 with HCl 1N aq. The mixture was extracted 3 times with ethyl acetate, dried over magnesium sulfate, filtered, evaporated and 4-[[1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-. 2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indrin-3-carbonyl] Amino] benzoic acid (87 mg, 50.6%) was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 758.4

４−（１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）安息香酸（４０ｍｇ、５２．８μｍｏｌ、当量：１）を、酢酸エチル（２５０μｌ）中でｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（３２．５ｍｇ、５２．８μｍｏｌ、当量：１；中間体２）、Ｎ−メチルモルホリン（５３．４ｍｇ、５８μｌ、５２８μｍｏｌ、当量：１０）及びＥｔＯＡｃ ５０％（１０１ｍｇ、９４．３μｌ、１５８μｍｏｌ、当量：３）中Ｔ３Ｐと室温で一晩撹拌した。飽和ＮａＨＣＯ３を添加し、混合物を酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−５％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［３−［［４−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］カルバモイル］フェニル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（２６ｍｇ、３６．３％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１３５５．７ c) tert-butyl (2R, 5S) -4- [2- [3-[[4-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxy) Carbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] carbamoyl] phenyl] carbamoyl]- 6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] ] Methyl] piperazin-1-carboxylate

4-(1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) ) -6- (4-Fluorobenzyl) -3-methylindrin-3-carboxamide) benzoic acid (40 mg, 52.8 μmol, equivalent: 1) in tert-butyl ((S)-) in ethyl acetate (250 μl). 1-(((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindolin-2-yl) -2-oxo-1- (tetrahydro-2H) -Pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (32.5 mg, 52.8 μmol, equivalent: 1; intermediate 2), N-methylmorpholin (53. The mixture was stirred overnight at room temperature with T3P in 4 mg, 58 μl, 528 μmol, equivalent: 10) and 50% EtOAc (101 mg, 94.3 μl, 158 μmol, equivalent: 3). Saturated NaHCO3 was added and the mixture was extracted 3 times with ethyl acetate, dried over magnesium sulphate, filtered and evaporated. The crude material was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-5% methanol) as eluent, and tert-butyl (2R, 5S) -4- [2- [3- [ [4-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl- Acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] carbamoyl] phenyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indrin-1- Il] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (26 mg, 36.3%) in white Obtained as a solid. MS: m / z (M + H) ⁺ = 1355.7

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（４−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）カルバモイル）フェニル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（２２ｍｇ、１６．２μｍｏｌ、当量：１）を、酢酸エチル（１０００μｌ）中でジオキサン４Ｍ（４００μｌ、１．６ｍｍｏｌ、当量：９８．６）中ＨＣｌと室温で一晩撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄した。固体を高減圧下で乾燥させ、Ｎ−（４−（（（Ｓ）−３−（（２，６−ジフルオロフェニル）カルバモイル）−２−（（Ｓ）−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−５−イル）カルバモイル）フェニル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド トリヒドロクロリド （１２．１ｍｇ、５９％）をオフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１１５５．６ d) N- [4-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino) propanoyl]] Amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] carbamoyl] phenyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R) , 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide trihydrochloride

(2R, 5S) -tert-butyl 4-(2-(3-((4-(((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl)) (Methyl) Amino) Propanamide) -2- (Tetrahydro-2H-Pyran-4-yl) Acetyl) -3-((2,6-difluorophenyl) Carbamoyl) Isoindrin-5-yl) Carbamoyl) Phenyl) Carbamoyl ) -6- (4-Fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxy The rate (22 mg, 16.2 μmol, equivalent: 1) was stirred overnight in ethyl acetate (1000 μl) with HCl in Dioxane 4M (400 μl, 1.6 mmol, equivalent: 98.6) at room temperature. The precipitate was filtered off and washed with diethyl ether. The solid was dried under high pressure and N-(4-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2-) (Methylamino) Propanamide) -2- (Tetrahydro-2H-pyran-4-yl) acetyl) Isoindrin-5-yl) Carbamoyl) Phenyl) -6- (4-Fluorobenzyl) -3-Methyl-1- (2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride (12.1 mg, 59%) was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 1155.6

１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（４０ｍｇ、６２．６μｍｏｌ、当量：１）を、酢酸エチル（３５０μｌ）中でｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−１−（（Ｓ）−７−アミノ−３−（（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）カルバモイル）−３，４−ジヒドロイソキノリン−２（１Ｈ）−イル）−３，３−ジメチル−１−オキソブタン−２−イル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（３８．８ｍｇ、６２．６μｍｏｌ、当量：１）（国際公開第２０１３／１９２２８６号に記載）、Ｎ−メチルモルホリン（６３．３ｍｇ、６８．８μｌ、６２６μｍｏｌ、当量：１０）及びＥｔＯＡｃ ５０％（１２０ｍｇ、１１２μｌ、１８８μｍｏｌ、当量：３）中Ｔ３Ｐと室温で一晩撹拌した。飽和ＮａＨＣＯ３を添加し、酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチルを使用したカラムクロマトグラフィーにより精製し、（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−３，３−ジメチルブタノイル）−３−（（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）カルバモイル）−１，２，３，４−テトラヒドロイソキノリン−７−イル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（４４．１ｍｇ、５６．８％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２４０．７２ Example 5
(3S) -2-((S) -3,3-dimethyl-2-((S) -2- (methylamino) propanamide) butanoyl) -7- (6- (4-fluorobenzyl) -3-3 Methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) -N- ((R) -1,2,3,4-tetrahydronaphthalene-1-yl) -1,2,3,4-tetrahydroisoquinolin-3-carboxamide trihydrochloride
a) tert-Butyl (2R, 5S) -4- [2- [3-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl)) Amino] propanoyl] amino] -3,3-dimethyl-butanoyl] -3-[[(1R) -tetraline-1-yl] carbamoyl] -3,4-dihydro-1H-isoquinolin-7-yl] carbamoyl]- 6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholine-4-yl] ] Methyl] Piperazine-1-carboxylate

1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6 -(4-Fluorobenzyl) -3-methylindrin-3-carboxylic acid (40 mg, 62.6 μmol, equivalent: 1) was added to tert-butyl ((S) -1-((((S) -1-) in ethyl acetate (350 μl). S) -1-((S) -7-amino-3-(((R) -1,2,3,4-tetrahydronaphthalene-1-yl) carbamoyl) -3,4-dihydroisoquinolin-2 (1H) ) -Il) -3,3-dimethyl-1-oxobutan-2-yl) amino) -1-oxopropan-2-yl) (methyl) carbamate (38.8 mg, 62.6 μmol, equivalent: 1) (international) Published No. 2013/192286), N-methylmorpholine (63.3 mg, 68.8 μl, 626 μmol, equivalent: 10) and EtOAc in 50% (120 mg, 112 μl, 188 μmol, equivalent: 3) with T3P at room temperature. Stirred evening. Saturated NaHCO3 was added, extracted 3 times with ethyl acetate, dried over magnesium sulfate, filtered and evaporated. The crude substance was applied to silica gel and purified by column chromatography using ethyl acetate as an eluent, and (2R, 5S) -tert-butyl 4-(2-(3-(((S) -2-((). S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -3,3-dimethylbutanoyl) -3-(((R) -1,2,3 , 4-Tetrahydronaphthalene-1-yl) carbamoyl) -1,2,3,4-tetrahydroisoquinolin-7-yl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl)- 2-Oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (44.1 mg, 56.8%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1240.72

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−３，３−ジメチルブタノイル）−３−（（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）カルバモイル）−１，２，３，４−テトラヒドロイソキノリン−７−イル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（３９ｍｇ、３１．４μｍｏｌ、当量：１）を、酢酸エチル（１ｍｌ）中でジオキサン４Ｍ（７００μｌ、２．８ｍｍｏｌ、当量：８９．１）中ＨＣｌと室温で一晩撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄した。固体を高減圧下で乾燥させ、（３Ｓ）−２−（（Ｓ）−３，３−ジメチル−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）ブタノイル）−７−（６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド）−Ｎ−（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）−１，２，３，４−テトラヒドロイソキノリン−３−カルボキサミド トリヒドロクロリド（３０ｍｇ、８３％）をオフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０４０．６２ b) (3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2- (methylamino) propanoyl] amino] butanoyl] -7-[[6-[(4-4-) Fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazin-1-yl] ] Acetyl] Indolin-3-carbonyl] Amino] -N-[(1R) -Tetraline-1-yl] -3,4-dihydro-1H-isoquinolin-3-carboxamide Trihydrochloride

(2R, 5S) -tert-butyl 4-(2-(3-(((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) ) Propanamide) -3,3-dimethylbutanoyl) -3-(((R) -1,2,3,4-tetrahydronaphthalene-1-yl) carbamoyl) -1,2,3,4-tetrahydroisoquinolin -7-Il) Carbamoyl) -6- (4-Fluorobenzyl) -3-Methylindrin-1-yl) -2-oxoethyl) -2-Methyl-5-(((R) -3-methylmorpholino) methyl ) Piperazin-1-carboxylate (39 mg, 31.4 μmol, equivalent: 1) stirred overnight at room temperature with HCl in dioxane 4M (700 μl, 2.8 mmol, equivalent: 89.1) in ethyl acetate (1 ml). bottom. The precipitate was filtered off and washed with diethyl ether. The solid was dried under high pressure and (3S) -2-((S) -3,3-dimethyl-2-((S) -2- (methylamino) propanamide) butanoyl) -7- (6-- (4-Fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) Indolin-3-carboxamide) -N-((R) -1,2,3,4-tetrahydronaphthalene-1-yl) -1,2,3,4-tetrahydroisoquinolin-3-carboxamide trihydrochloride (30 mg,) 83%) was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 1040.62

２−（１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（４０ｍｇ、５７．５μｍｏｌ、当量：１）を、酢酸エチル（３５０μｌ）中でｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−１−（（Ｓ）−７−アミノ−３−（（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）カルバモイル）−３，４−ジヒドロイソキノリン−２（１Ｈ）−イル）−３，３−ジメチル−１−オキソブタン−２−イル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（３５．６ｍｇ、５７．５μｍｏｌ、当量：１）、Ｎ−メチルモルホリン（５８．１ｍｇ、６３．２μｌ、５７５μｍｏｌ、当量：１０）及びＥｔＯＡｃ ５０％（１１０ｍｇ、１０３μｌ、１７２μｍｏｌ、当量：３）中Ｔ３Ｐと室温で一晩撹拌した。飽和ＮａＨＣＯ３溶液を添加し、酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチルを使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［３−［［２−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−３，３−ジメチル−ブタノイル］−３−［［（１Ｒ）−テトラリン−１−イル］カルバモイル］−３，４−ジヒドロ−１Ｈ−イソキノリン−７−イル］アミノ］−２−オキソ−エチル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（４０ｍｇ、５３．６％）をオフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２９７．７４ Example 6
(3S) -2-((S) -3,3-dimethyl-2-((S) -2- (methylamino) propanoamide) butanoyl) -7- (2- (6- (4-fluorobenzyl)) -3-Methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) Acetamide) -N-((R) -1,2,3,4-tetrahydronaphthalene-1-yl) -1,2,3,4-tetrahydroisoquinolin-3-carboxamide trihydrochloride
a) tert-Butyl (2R, 5S) -4- [2- [3-[[2-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxy Carbonyl (methyl) amino] propanoyl] amino] -3,3-dimethyl-butanoyl] -3-[[(1R) -tetraline-1-yl] carbamoyl] -3,4-dihydro-1H-isoquinolin-7-yl ] Amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indrin-1-yl] -2-oxo-ethyl] -2-methyl-5-[ [(3R) -3-methylmorpholine-4-yl] methyl] piperazin-1-carboxylate

2-(1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) ) -6- (4-Fluorobenzyl) -3-methylindrin-3-carboxamide) acetic acid (40 mg, 57.5 μmol, equivalent: 1) in ethyl acetate (350 μl) with tert-butyl ((S) -1). -(((S) -1-((S) -7-amino-3-(((R) -1,2,3,4-tetrahydronaphthalene-1-yl) carbamoyl) -3,4-dihydroisoquinoline) -2 (1H) -yl) -3,3-dimethyl-1-oxobutan-2-yl) amino) -1-oxopropan-2-yl) (methyl) carbamate (35.6 mg, 57.5 μmol, equivalent: 1), N-methylmorpholine (58.1 mg, 63.2 μl, 575 μmol, equivalent: 10) and EtOAc in 50% (110 mg, 103 μl, 172 μmol, equivalent: 3) were stirred with T3P overnight at room temperature. Saturated NaHCO3 solution was added, extracted 3 times with ethyl acetate, dried over magnesium sulphate, filtered and evaporated. The crude substance was applied to silica gel, purified by column chromatography using ethyl acetate as an eluent, and tert-butyl (2R, 5S) -4- [2- [3-[[2-[[(3S)-)-. 2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -3,3-dimethyl-butanoyl] -3-[[(1R) -tetraline -1-yl] carbamoyl] -3,4-dihydro-1H-isoquinoline-7-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl- Indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazin-1-carboxylate (40 mg, 53.6%) ) Was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 1297.74

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−３，３−ジメチルブタノイル）−３−（（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）カルバモイル）−１，２，３，４−テトラヒドロイソキノリン−７−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（３６ｍｇ、２７．７μｍｏｌ、当量：１）を酢酸エチル（８００μＬ）中でジオキサン４Ｍ（６５０μｌ、２．６ｍｍｏｌ、当量：９３．７）中ＨＣｌと室温で一晩撹拌した。沈殿した固形物を濾別し、ジエチルエーテルで洗浄した。固体を高減圧下で乾燥させ、（３Ｓ）−２−（（Ｓ）−３，３−ジメチル−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）ブタノイル）−７−（２−（６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド）アセトアミド）−Ｎ−（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）−１，２，３，４−テトラヒドロイソキノリン−３−カルボキサミド トリヒドロクロリド （２４．５ｍｇ、７３．２％）をオフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０９７．６３ b) (3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2- (methylamino) propanoyl] amino] butanoyl] -7-[[2-[[6-- [(4-Fluorophenyl) Methyl] -3-Methyl-1- [2-[(2R, 5R) -5-Methyl-2-[[(3R) -3-Methylmorpholine-4-yl] Methyl] Piperazine] -1-yl] acetyl] indolin-3-carbonyl] amino] acetyl] amino] -N-[(1R) -tetraline-1-yl] -3,4-dihydro-1H-isoquinolin-3-carboxamide trihydrochloride

(2R, 5S) -tert-butyl 4-(2-(3-((2-(((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl)) (Methyl) amino) propanamide) -3,3-dimethylbutanoyl) -3-(((R) -1,2,3,4-tetrahydronaphthalene-1-yl) carbamoyl) -1,2,3 4-Tetrahydroisoquinoline-7-yl) amino) -2-oxoethyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindolin-1-yl) -2-oxoethyl) -2-methyl-5-( ((R) -3-Methylmorpholino) methyl) piperazin-1-carboxylate (36 mg, 27.7 μmol, equivalent: 1) in ethyl acetate (800 μL) with dioxane 4M (650 μl, 2.6 mmol, equivalent: 93. 7) Stirred overnight with medium HCl at room temperature. The precipitated solid was filtered off and washed with diethyl ether. The solid was dried under high pressure and (3S) -2-((S) -3,3-dimethyl-2-((S) -2- (methylamino) propanoamide) butanoyl) -7- (2-). (6- (4-Fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) ) Acetyl) Indolin-3-carboxamide) Acetamide) -N-((R) -1,2,3,4-tetrahydronaphthalene-1-yl) -1,2,3,4-tetrahydroisoquinolin-3-carboxamide tri Hydrochloride (24.5 mg, 73.2%) was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 1097.63

（ｄｐｐｆ）ＰｄＣｌ_２．ＣＨ_２Ｃｌ_２（２２．４ｍｇ、２７．４μｍｏｌ、当量：０．０１）と、ＴＨＦ（１１ｍｌ、５．４８ｍｍｏｌ、当量：２）中（４−フルオロベンジル）塩化亜鉛（ＩＩ）０．５Ｍの溶液とを、ＴＨＦ（１０ｍｌ）中ｔｅｒｔ−ブチル ６−ブロモ−３−（２−シアノエチル）−３−メチルインドリン−１−カルボキシレート（１０００ｍｇ、２．７４ｍｍｏｌ、当量：１）（国際公開第２００９１５８０１１号に記載）の明黄色の溶液に添加した。シールド管を密閉し、暗褐色の懸濁液を９０℃に加熱し、２時間撹拌した。水を数滴添加した後、粗混合物をシリカゲルに適用し、溶出剤としてヘプタン／酢酸エチル（０−３０％酢酸エチル）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル ３−（２−シアノエチル）−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−カルボキシレート（１．００３ｇ、９２．９％）を明黄色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ−ＢＯＣ＋Ｈ）^＋＝２９５．２ Example 7
N1-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-) 2H-pyran-4-yl) acetyl) isoindolin-5-yl) -N3- (3- (6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5) -Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-yl) propyl) malonamide trihydrochloride
a) tert-Butyl 3- (2-cyanoethyl) -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-carboxylate

(Dppf) PdCl ₂ . A solution of _{CH 2 Cl 2} (22.4 mg, 27.4 μmol, equivalent: 0.01) and zinc (4-fluorobenzyl) zinc chloride (II) 0.5 M in THF (11 ml, 5.48 mmol, equivalent: 2). In THF (10 ml), tert-butyl 6-bromo-3- (2-cyanoethyl) -3-methylindrin-1-carboxylate (1000 mg, 2.74 mmol, equivalent: 1) (International Publication No. 2009158011). It was added to the bright yellow solution (described). The shield tube was sealed and the dark brown suspension was heated to 90 ° C. and stirred for 2 hours. After adding a few drops of water, the crude mixture was applied to silica gel and purified by column chromatography using heptane / ethyl acetate (0-30% ethyl acetate) as eluent and tert-butyl 3- (2-cyanoethyl). ) -6-[(4-Fluorophenyl) methyl] -3-methyl-indolin-1-carboxylate (1.003 g, 92.9%) was obtained as a bright yellow solid. MS: m / z (M-BOC + H) ⁺ = 295.2

ジオキサン（１０．６ｍｌ、４２．６ｍｍｏｌ、当量：１６）中ＨＣｌ ４Ｍを、メタノール（１０ｍｌ）中ｔｅｒｔ−ブチル ３−（２−シアノエチル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−カルボキシレート（１．０５０ｇ、２．６６ｍｍｏｌ、当量：１）の明黄色の懸濁液に室温で添加した。黄色の溶液を室温で２時間撹拌した。溶媒をエバポレートし、黄色の油を酢酸エチルで希釈し、飽和ＮａＨＣＯ_３水溶液で抽出した。有機層を硫酸マグネシウムで乾燥させ、濾過し、エバポレートして、３−［６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−イル］プロパンニトリル（７６３ｍｇ、９７．４％）をオレンジ色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝２９５．２ b) 3- [6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-yl] propanenitrile

HCl 4M in dioxane (10.6 ml, 42.6 mmol, equivalent: 16), tert-butyl 3- (2-cyanoethyl) -6- (4-fluorobenzyl) -3-methylindoline-1 in methanol (10 ml). -Carboxylate (1.050 g, 2.66 mmol, eq: 1) was added to a bright yellow suspension at room temperature. The yellow solution was stirred at room temperature for 2 hours. The solvent was evaporated, the yellow oil was diluted with ethyl acetate and extracted with _{saturated aqueous NaHCO 3 solution.} The organic layer is dried over magnesium sulphate, filtered, evaporated and 3- [6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-yl] propanenitrile (763 mg, 97.4%). ) Was obtained as an orange oil. MS: m / z (M + H) ⁺ = 295.2

３−（６−（４−フルオロベンジル）−３−メチルインドリン−３−イル）プロパンニトリル（７６３ｍｇ、２．５９ｍｍｏｌ、当量：１）を酢酸エチル（１０ｍｌ）に溶解し、２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）酢酸（９６３ｍｇ、２．５９ｍｍｏｌ、当量：１）、ＥｔＯＡｃ ５０％（４．９５ｇ、４．５８ｍｌ、７．７８ｍｍｏｌ、当量：３）中Ｔ３Ｐ及びＮ−メチルモルホリン（２．６２ｇ、２．８５ｍｌ、２５．９ｍｍｏｌ、当量：１０）を添加し、反応物を室温で一晩撹拌した。反応混合物を酢酸エチルで希釈し、飽和ＮａＨＣＯ_３で洗浄し、Ｍｇ_２ＳＯ４で乾燥させ、真空中で濃縮した。粗残留物をシリカゲルに適用し、溶出剤として酢酸エチル／ヘプタン（５０−１００％酢酸エチル）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［３−（２−シアノエチル）−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（９８３ｍｇ、５８．５％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６４８．４ c) tert-Butyl (2R, 5S) -4- [2- [3- (2-cyanoethyl) -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2- Oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazine-1-carboxylate

3- (6- (4-Fluorobenzyl) -3-methylindolin-3-yl) propanenitrile (763 mg, 2.59 mmol, equivalent: 1) was dissolved in ethyl acetate (10 ml) and 2-((2S, 2S,). 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetate (963 mg, 2.59 mmol, equivalent: 1), Add T3P and N-methylmorpholine (2.62 g, 2.85 ml, 25.9 mmol, equivalent: 10) in 50% of EtOAc (4.95 g, 4.58 ml, 7.78 mmol, equivalent: 3) to the reaction product. Was stirred overnight at room temperature. The reaction mixture was diluted with ethyl acetate _{, washed with saturated NaHCO 3} , dried over Mg ₂ SO4 and concentrated in vacuo. The crude residue was applied to silica gel and purified by column chromatography using ethyl acetate / heptane (50-100% ethyl acetate) as eluent and tert-butyl (2R, 5S) -4- [2- [3]. -(2-Cyanoethyl) -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3 -Methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (983 mg, 58.5%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 648.4

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（２−シアノエチル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（８５０ｍｇ、１．３１ｍｍｏｌ、当量：１）をＭｅＯＨ ７Ｎ（１５．７ｇ、２０ｍｌ、１４０ｍｍｏｌ、当量：１０７）中アンモニアに溶解し、スパチュラを用いてラネーニッケル（１．３１ｍｍｏｌ、当量：１）を触媒量で添加した。反応器を脱気し、水素でパージし（３回）、反応物を５０℃に加熱し、水素雰囲気下で８時間激しく撹拌した。反応物を室温に一晩冷ました。反応混合物をデカライト（ｄｅｃａｌｉｔｅ）で濾過し、メタノールで洗浄してエバポレートした。粗残留物をシリカゲルに適用し、溶出剤としてジクロロメタン／メタノール（０−５％メタノールを使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［３−（３−アミノプロピル）−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（４３６ｍｇ、５１％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６５２．４ d) tert-Butyl (2R, 5S) -4- [2- [3- (3-aminopropyl) -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2 -Oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazine-1-carboxylate

(2R, 5S) -tert-butyl 4-(2- (3- (2-cyanoethyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl -5-(((R) -3-methylmorpholino) methyl) piperazine-1-carboxylate (850 mg, 1.31 mmol, equivalent: 1) in MeOH 7N (15.7 g, 20 ml, 140 mmol, equivalent: 107) It was dissolved in ammonia and lane nickel (1.31 mmol, equivalent: 1) was added in catalytic amounts using a spatula. The reactor was degassed, purged with hydrogen (3 times), the reaction was heated to 50 ° C. and vigorously stirred in a hydrogen atmosphere for 8 hours. The reaction was cooled to room temperature overnight. The reaction mixture was filtered through decalite, washed with methanol and evaporated. The crude residue was applied to silica gel, purified by column chromatography using dichloromethane / methanol (0-5% methanol as eluent), and tert-butyl (2R, 5S) -4- [2- [3- (3). −Aminopropyl) -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methyl Morphorin-4-yl] methyl] piperazin-1-carboxylate (436 mg, 51%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 652.4.

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（３−アミノプロピル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（５０ｍｇ、７６．７μｍｏｌ、当量：１）、３−（ベンジルオキシ）−３−オキソプロパン酸（１７．２ｍｇ、８４．４μｍｏｌ、当量：１．１）、ＨＡＴＵ（３２．１ｍｇ、８４．４μｍｏｌ、当量：１．１）及びＤＩＰＥＡ（１９．８ｍｇ、２６．８μｌ、１５３μｍｏｌ、当量：２）をＤＭＦ（１ｍｌ）に溶解し、明黄色の溶液を室温で一晩撹拌した。酢酸エチルを添加し、混合物を水及びブラインで抽出した。粗残留物をシリカゲルに適用し、溶出剤として酢酸エチル／ヘプタン（０−１００％酢酸エチル）を使用したカラムクロマトグラフィー（塩基性シリカ）により精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［３−［３−［（３−ベンジルオキシ−３−オキソ−プロパノイル）アミノ］プロピル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（４８ｍｇ、７５．６％）を無色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝８２８．５ e) tert-butyl (2R, 5S) -4- [2- [3- [3-[(3-benzyloxy-3-oxo-propanoyl) amino] propyl] -6-[(4-fluorophenyl) methyl ] -3-Methyl-Indoline-1-yl] -2-oxo-ethyl] -2-Methyl-5-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazine-1-carboxylate

(2R, 5S) -tert-butyl 4-(2- (3- (3-aminopropyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2- Methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (50 mg, 76.7 μmol, equivalent: 1), 3- (benzyloxy) -3-oxopropanoic acid (17. 2 mg, 84.4 μmol, equivalent: 1.1), HATU (32.1 mg, 84.4 μmol, equivalent: 1.1) and DIPEA (19.8 mg, 26.8 μl, 153 μmol, equivalent: 2) in DMF (1 ml). ), And the bright yellow solution was stirred at room temperature overnight. Ethyl acetate was added and the mixture was extracted with water and brine. The crude residue was applied to silica gel, purified by column chromatography (basic silica) using ethyl acetate / heptan (0-100% ethyl acetate) as eluent, and tert-butyl (2R, 5S) -4-. [2- [3- [3-[(3-benzyloxy-3-oxo-propanoyl) amino] propyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl]- 2-Oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (48 mg, 75.6%) is obtained as a colorless oil. rice field. MS: m / z (M + H) ⁺ = 828.5

Ｐｄ−Ｃ（１４．５ｍｇ、１３．６μｍｏｌ、当量：０．２５）を、メタノール（０．５ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（３−（３−（ベンジルオキシ）−３−オキソプロパンアミド）プロピル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（４５ｍｇ、５４．３μｍｏｌ、当量：１）の透明な溶液に添加し、反応物を水素雰囲気下室温で６時間撹拌した。反応混合物をデカライトで濾過し、メタノールで洗浄した。濾液をエバポレートし、３−［３−［１−［２−［（２Ｓ，５Ｒ）−４−ｔｅｒｔ−ブトキシカルボニル−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−イル］プロピルアミノ］−３−オキソ−プロパン酸（３３ｍｇ、８２．３％）を明黄色の泡として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝７３８．４ f) 3- [3- [1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl]] Piperazine-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-yl] propylamino] -3-oxo-propanoic acid

Pd-C (14.5 mg, 13.6 μmol, equivalent: 0.25) in methanol (0.5 ml) (2R, 5S) -tert-butyl 4- (2- (3- (3- (3- (3- (3-) 3- (3-) (Benzyloxy) -3-oxopropanamide) propyl) -6- (4-fluorobenzyl) -3-methylindolin-1-yl) -2-oxoethyl) -2-methyl-5-(((R)-)- It was added to a clear solution of 3-methylmorpholino) methyl) piperazin-1-carboxylate (45 mg, 54.3 μmol, equivalent: 1) and the reaction was stirred under a hydrogen atmosphere at room temperature for 6 hours. The reaction mixture was filtered through Decalite and washed with methanol. The filtrate was evaporated and 3-[3- [1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl) ] Methyl] Piperazine-1-yl] Acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-Indoline-3-yl] Propylamino] -3-oxo-propanoic acid (33 mg, 82.3) %) Was obtained as a bright yellow foam. MS: m / z (M + H) ⁺ = 738.4

３−（（３−（１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−イル）プロピル）アミノ）−３−オキソプロパン酸（３０ｍｇ、４０．７μｍｏｌ、当量：１）、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（２５ｍｇ、４０．７μｍｏｌ、当量：１；中間体２）、ＥｔＯＡｃ（７７．６ｍｇ、７２．６μｌ、１２２μｍｏｌ、当量：３）中Ｔ３Ｐ ５０％及びＮ−メチルモルホリン（４１．１ｍｇ、４４．７μｌ、４０７μｍｏｌ、当量：１０）を合わせて酢酸エチル（０．３ｍｌ）に溶解し、黄色の溶液を室温で一晩撹拌した。溶媒をエバポレートし、粗残留物をシリカゲルに適用し、酢酸エチル／メタノール（０−１０％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［３−［３−［［３−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］アミノ］−３−オキソ−プロパノイル］アミノ］プロピル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（１３ｍｇ、２３．９％）を黄色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１３３５．７ g) tert-butyl (2R, 5S) -4- [2- [3- [3-] [3-[[(3S) -2-[(2S) -2-[[(2S) -2-[ tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindolin-5-yl] amino] -3 -Oxo-propanoyl] amino] propyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indoline-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) ) -3-Methylmorpholin-4-yl] methyl] piperazin-1-carboxylate

3-((3- (1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-yl) propyl) amino) -3-oxopropanoic acid (30 mg, 40.7 μmol, equivalent: 1), tert-butyl ( (S) -1-(((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindrin-2-yl) -2-oxo-1- (Tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (25 mg, 40.7 μmol, equivalent: 1; intermediate 2), EtOAc (77.6 mg , 72.6 μl, 122 μmol, equivalent: 3) 50% of T3P and N-methylmorpholin (41.1 mg, 44.7 μl, 407 μmol, equivalent: 10) were combined and dissolved in ethyl acetate (0.3 ml), and yellow. The solution was stirred at room temperature overnight. The solvent was evaporated, the crude residue was applied to silica gel and purified by column chromatography using ethyl acetate / methanol (0-10% methanol) and tert-butyl (2R, 5S) -4- [2-[ 3- [3-[[3-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydro Piran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindolin-5-yl] amino] -3-oxo-propanoyl] amino] propyl] -6-[(4-fluoro Phenyl) Methyl] -3-methyl-Indoline-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1- Carboxylate (13 mg, 23.9%) was obtained as a yellow solid. MS: m / z (M + H) ⁺ = 1335.7

ジオキサン（２４．６ｍｇ、２０．５μｌ、６７４μｍｏｌ、当量：１００）中ＨＣｌ ４Ｎを、酢酸エチル（０．５ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（３−（３−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−３−オキソプロパンアミド）プロピル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（９ｍｇ、６．７４μｍｏｌ、当量：１）の黄色の溶液に添加し、反応物を室温で一晩撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄し、真空中で乾燥させて、Ｎ１−（（Ｓ）−３−（（２，６−ジフルオロフェニル）カルバモイル）−２−（（Ｓ）−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−５−イル）−Ｎ３−（３−（６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−イル）プロピル）マロンアミド トリヒドロクロリド （５．６ｍｇ、６６．８％）を明黄色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１１３５．６ h) N'-[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino) propanoyl] amino]- 2-Tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] -N- [3- [6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 2R,) 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-yl] propyl] propandiamide trihydrochloride

HCl 4N in dioxane (24.6 mg, 20.5 μl, 674 μmol, equivalent: 100) and (2R, 5S) -tert-butyl 4- (2- (3- (3- (3- (3-)) in ethyl acetate (0.5 ml). 3-(((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4) -Il) acetyl) -3-((2,6-difluorophenyl) carbamoyl) isoindrin-5-yl) amino) -3-oxopropanamide) propyl) -6- (4-fluorobenzyl) -3-methyl Indoline-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (9 mg, 6.74 μmol, equivalent: 1) yellow Was added to the solution and the reaction was stirred at room temperature overnight. The precipitate was filtered off, washed with diethyl ether, dried in vacuo and N1-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-). ((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) -N3- (3- (6- (4-fluoro) Benzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-3 Il) Propyl) malonamide trihydrochloride (5.6 mg, 66.8%) was obtained as a bright yellow solid. MS: m / z (M + H) ⁺ = 1135.6

２−（（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（１４０ｍｇ、２０１μｍｏｌ、当量：１）を、酢酸エチル（１．１ｍｌ）中でｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（１２４ｍｇ、２０１μｍｏｌ、当量：１；中間体２）、Ｎ−メチルモルホリン（２０４ｍｇ、２２１μｌ、２．０１ｍｍｏｌ、当量：１０）及びＥｔＯＡｃ ５０％（３８４ｍｇ、３５９μｌ、６０４μｍｏｌ、当量：３）中Ｔ３Ｐと室温で３時間撹拌した。飽和ＮａＨＣＯ３を添加し、酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−１０％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｓ）−３−［［２−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］アミノ］−２−オキソ−エチル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（１９５ｍｇ、７４．９％）を明黄色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２９３．７ Example 8
(3S) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino)) Propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1 -[2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide trihydrochloride a) tert-butyl (2R, 5S) -4- [2-[(3S) -3-[[2-[[(3S) -2-[(2S) -2-[[(2S) -2-] [Tert-Butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino]- 2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R)) -3-Methylmorpholin-4-yl] Methyl] piperazin-1-carboxylate

2-((S) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxamide) tert-butyl ((140 mg, 201 μmol, equivalent: 1) in ethyl acetate (1.1 ml). S) -1-(((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindrin-2-yl) -2-oxo-1-( Tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (124 mg, 201 μmol, equivalent: 1; intermediate 2), N-methylmorpholin (204 mg, 221 μl) , 2.01 mmol, equivalent: 10) and 50% EtOAc (384 mg, 359 μl, 604 μmol, equivalent: 3) with T3P and stirred at room temperature for 3 hours. Saturated NaHCO3 was added, extracted 3 times with ethyl acetate, dried over magnesium sulfate, filtered and evaporated. The crude material was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-10% methanol) as eluent, and tert-butyl (2R, 5S) -4- [2-[(3S)). -3-[[2-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran- 4-Il-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-Methyl-Indoline-1-yl] -2-oxo-ethyl] -2-Methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (195 mg) , 74.9%) was obtained as a bright yellow solid. MS: m / z (M + H) ⁺ = 1293.7

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（１８８ｍｇ、１４５μｍｏｌ、当量：１）を、酢酸エチル（３ｍｌ）中でジオキサン４Ｍ（３ｍｌ、１２ｍｍｏｌ、当量：８２．６）中ＨＣｌと室温で一晩撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄し、高減圧下で乾燥させて、（３Ｓ）−Ｎ−［２−［［（３Ｓ）−３−［（２，６−ジフルオロフェニル）カルバモイル］−２−［（２Ｓ）−２−［［（２Ｓ）−２−（メチルアミノ）プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］イソインドリン−５−イル］アミノ］−２−オキソ−エチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−１−［２−［（２Ｒ，５Ｒ）−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］インドリン−３−カルボキサミド トリヒドロクロリド（１６１ｍｇ、９２．１％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０９３．５ b) (3S) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methyl) Amino) propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl -1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide tri Hydrochloride

(2R, 5S) -tert-butyl 4-(2-((S) -3-((2-(((S) -2-((S) -2-((S) -2-((tert) -Butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) -3-((2,6-difluorophenyl) carbamoyl) isoindrin-5-yl) amino ) -2-oxoethyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) Methyl) piperazin-1-carboxylate (188 mg, 145 μmol, equivalent: 1) was stirred overnight with HCl in dioxane 4M (3 ml, 12 mmol, equivalent: 82.6) in ethyl acetate (3 ml) at room temperature. The precipitate was filtered off, washed with diethyl ether, dried under high vacuum and (3S) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl]-. 2-[(2S) -2-[[(2S) -2- (methylamino) propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] amino] -2-oxo -Ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4 -Methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide trihydrochloride (161 mg, 92.1%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1093.5

ラセミ体のｔｅｒｔ−ブチル−メチル ６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１，３−ジカルボキシレート（１．２１ｇ、３．０３ｍｍｏｌ、当量：１）をキラル分離し、（Ｒ）−１−ｔｅｒｔ−ブチル ３−メチル ６−（４−フルオロベンジル）−３−メチルインドリン−１，３−ジカルボキシレート（５７１ｍｇ、４７．２％）を無色の油として得た。ＭＳ：ｍ／ｚ Ｍ^＋＝３９９．２０ Example 9
(3R) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino)) Propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1 -[2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide trihydrochloride a) (R) -1-tert-butyl 3-methyl 6- (4-fluorobenzyl) -3-methylindrin-1,3-dicarboxylate

The tert-butyl-methyl 6-[(4-fluorophenyl) methyl] -3-methyl-indrin-1,3-dicarboxylate (1.21 g, 3.03 mmol, equivalent: 1) of the racemic form was chirally separated. , (R) -1-tert-Butyl 3-methyl 6- (4-fluorobenzyl) -3-methylindrin-1,3-dicarboxylate (571 mg, 47.2%) was obtained as a colorless oil. MS: m / z M ⁺ = 399.20

（Ｒ）−１−ｔｅｒｔ−ブチル ３−メチル ６−（４−フルオロベンジル）−３−メチルインドリン−１，３−ジカルボキシレートを使用して、実施例１ｃ）と同じ方法で生成物を調製し、メチル（３Ｒ）−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボキシレート（３７３ｍｇ、９５．４％）を明黄色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝３００．１４ b) Methyl (3R) -6-[(4-fluorophenyl) methyl] -3-methyl-indoline-3-carboxylate

(R) -1-tert-Butyl 3-methyl 6- (4-fluorobenzyl) -3-methyl indoline-1,3-dicarboxylate was used to prepare the product in the same manner as in Example 1c). Then, methyl (3R) -6-[(4-fluorophenyl) methyl] -3-methyl-indoline-3-carboxylate (373 mg, 95.4%) was obtained as a bright yellow solid. MS: m / z (M + H) ⁺ = 30.14

（Ｒ）−メチル ６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキシレート（３１４ｍｇ、１．０５ｍｍｏｌ、当量：１）及び２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）酢酸（３９０ｍｇ、１．０５ｍｍｏｌ、当量：１）を出発物質として使用して、実施例１ｄ）と同じ方法で生成物を調製し、メチル（３Ｒ）−１−［２−［（２Ｓ，５Ｒ）−４−ｔｅｒｔ−ブトキシカルボニル−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボキシレート（５０５ｍｇ、７３．７％）を明黄色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６５３．３７ c) (3R) -1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazine- 1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carboxylate

(R) -Methyl 6- (4-fluorobenzyl) -3-methylindrin-3-carboxylate (314 mg, 1.05 mmol, equivalent: 1) and 2-((2S, 5R) -4- (tert-butoxy) Examples using carbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetate (390 mg, 1.05 mmol, equivalent: 1) as a starting material. The product was prepared in the same manner as in 1d) and methyl (3R) -1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-]. Methylmorpholin-4-yl] methyl] piperazine-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carboxylate (505 mg, 73.7%) Obtained as yellow oil. MS: m / z (M + H) ⁺ = 653.37

（Ｒ）−メチル １−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキシレート（５００ｍｇ、７６６μｍｏｌ、当量：１）を出発物質として使用して、実施例１ｅ）と同じ方法で生成物を調製し、（Ｒ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（４４１ｍｇ、６９０μｍｏｌ、９０．１％）をオフホワイトの泡として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６３９．３６ (R) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazine-1-yl) Acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylic acid

(R) -Methyl 1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) ) Acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylate (500 mg, 766 μmol, equivalent: 1) as a starting material to produce the product in the same manner as in Example 1e). Prepared (R) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylic acid (441 mg, 690 μmol, 90.1%) was obtained as an off-white foam. MS: m / z (M + H) ⁺ = 639.36

（Ｒ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（４３０ｍｇ、６７３μｍｏｌ、当量：１）を出発物質として使用して、実施例２ａ）と同じ方法で生成物を調製し、（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｒ）−６−（４−フルオロベンジル）−３−（（２−メトキシ−２−オキソエチル）カルバモイル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（４５５ｍｇ、６４１μｍｏｌ、９５．２％）を明黄色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝７１０．４ d) (2R, 5S) -tert-butyl 4-(2-((R) -6- (4-fluorobenzyl) -3-((2-methoxy-2-oxoethyl) carbamoyl) -3-methylindoline- 1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazine-1-carboxylate

(R) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) The product was prepared in the same manner as in Example 2a) using acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylic acid (430 mg, 673 μmol, equivalent: 1) as a starting material. Then, (2R, 5S) -tert-butyl 4-(2-((R) -6- (4-fluorobenzyl) -3-((2-methoxy-2-oxoethyl) carbamoyl) -3-methylindrin- 1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (455 mg, 641 μmol, 95.2%) in a bright yellow oil Got as. MS: m / z (M + H) ⁺ = 710.4

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｒ）−６−（４−フルオロベンジル）−３−（（２−メトキシ−２−オキソエチル）カルバモイル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（４３５ｍｇ、６１３μｍｏｌ、当量：１）を出発物質として使用して、実施例２ｂ）と同じ方法で生成物を調製し、２−［［（３Ｒ）−１−［２−［（２Ｓ，５Ｒ）−４−ｔｅｒｔ−ブトキシカルボニル−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボニル］アミノ］酢酸 （２８３ｍｇ、６６．４％）を明黄色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６９６．３７９３ e) 2-[[(3R) -1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl]] Methyl] piperazine-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carbonyl] amino] acetic acid

(2R, 5S) -tert-butyl 4-(2-((R) -6- (4-fluorobenzyl) -3-((2-methoxy-2-oxoethyl) carbamoyl) -3-methylindrin-1- Il) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (435 mg, 613 μmol, equivalent: 1) was used as a starting material. The product was prepared in the same manner as in Example 2b) and 2-[[(3R) -1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[( 3R) -3-methylmorpholin-4-yl] methyl] piperazine-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indrin-3-carbonyl] amino] acetic acid (283 mg) , 66.4%) was obtained as a bright yellow solid. MS: m / z (M + H) ⁺ = 696.3793

２−（（Ｒ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（１４０ｍｇ、２０１μｍｏｌ、当量：１）を、酢酸エチル（１．１ｍｌ）中でｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（１２４ｍｇ、２０１μｍｏｌ、当量：１；中間体２）、Ｎ−メチルモルホリン（２０４ｍｇ、２２１μｌ、２．０１ｍｍｏｌ、当量：１０）及びＥｔＯＡｃ ５０％（３８４ｍｇ、３５９μｌ、６０４μｍｏｌ、当量：３）中Ｔ３Ｐと室温で一晩撹拌した。飽和ＮａＨＣＯ３溶液を添加し、酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−１０％メタノール）を使用したカラムクロマトグラフィーにより精製し、（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｒ）−３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（１７７ｍｇ、６８％）を明黄色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２９３．６６ f) tert-butyl (2R, 5S) -4- [2-[(3R) -3-[[2-[[(3S) -2-[(2S) -2-[[(2S) -2-] [Tert-Butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino]- 2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R)) -3-Methylmorpholin-4-yl] Methyl] piperazin-1-carboxylate

2-((R) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxamide) tert-butyl ((140 mg, 201 μmol, equivalent: 1) in ethyl acetate (1.1 ml). S) -1-(((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindrin-2-yl) -2-oxo-1-( Tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (124 mg, 201 μmol, equivalent: 1; intermediate 2), N-methylmorpholin (204 mg, 221 μl) , 2.01 mmol, equivalent: 10) and 50% EtOAc (384 mg, 359 μl, 604 μmol, equivalent: 3) with T3P and stirred overnight at room temperature. Saturated NaHCO3 solution was added, extracted 3 times with ethyl acetate, dried over magnesium sulphate, filtered and evaporated. The crude material was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-10% methanol) as eluent, and (2R, 5S) -tert-butyl 4-(2-((R)). -3-((2-(((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-) 2H-pyran-4-yl) acetyl) -3-((2,6-difluorophenyl) carbamoyl) isoindolin-5-yl) amino) -2-oxoethyl) carbamoyl) -6- (4-fluorobenzyl)- 3-Methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (177 mg, 68%) in bright yellow Obtained as a solid. MS: m / z (M + H) ⁺ = 1293.66

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｒ）−３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（１７８ｍｇ、１３８μｍｏｌ、当量：１）を、酢酸エチル（３ｍｌ）中でジオキサン４Ｍ（３ｍｌ、１２ｍｍｏｌ、当量：８７．２）中ＨＣｌと室温で一晩撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄した。高減圧下で固体を乾燥させ、（３Ｒ）−Ｎ−［２−［［（３Ｓ）−３−［（２，６−ジフルオロフェニル）カルバモイル］−２−［（２Ｓ）−２−［［（２Ｓ）−２−（メチルアミノ）プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］イソインドリン−５−イル］アミノ］−２−オキソ−エチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−１−［２−［（２Ｒ，５Ｒ）−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］インドリン−３−カルボキサミド トリヒドロクロリド（１４３ｍｇ、８６．４％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０９３．５５ g) (3R) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methyl) Amino) propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl -1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazine-1-yl] acetyl] indolin-3-carboxamide tri Hydrochloride

(2R, 5S) -tert-butyl 4-(2-((R) -3-((2-(((S) -2-((S) -2-((S) -2-((tert) -Butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) -3-((2,6-difluorophenyl) carbamoyl) isoindrin-5-yl) amino ) -2-oxoethyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) Methyl) piperazin-1-carboxylate (178 mg, 138 μmol, equivalent: 1) was stirred overnight with HCl in dioxane 4M (3 ml, 12 mmol, equivalent: 87.2) in ethyl acetate (3 ml) at room temperature. The precipitate was filtered off and washed with diethyl ether. The solid was dried under high vacuum and (3R) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[( 2S) -2- (methylamino) propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindoline-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) ) Methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl ] Indoline-3-carboxamide trihydrochloride (143 mg, 86.4%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1093.55

ラセミ体のｔｅｒｔ−ブチル−メチル ６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１，３−ジカルボキシレート（１．２１ｇ、３．０３ｍｍｏｌ、当量：１）をキラル分離し、（Ｓ）−１−ｔｅｒｔ−ブチル ３−メチル ６−（４−フルオロベンジル）−３−メチルインドリン−１，３−ジカルボキシレート（５５５ｍｇ、４５．９％）を無色の油として得た。ＭＳ：ｍ／ｚ Ｍ＋＝３９９．２ Example 10
(3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2- (methylamino) propanoyl] amino] butanoyl] -7-[[2-[[(3S)- 6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] ] Piperazin-1-yl] acetyl] indolin-3-carbonyl] amino] acetyl] amino] -N-[(1R) -tetraline-1-yl] -3,4-dihydro-1H-isoquinolin-3-carboxamide tri Hydrochloride a) (S) -1-tert-butyl 3-methyl 6- (4-fluorobenzyl) -3-methylindrin-1,3-dicarboxylate

The tert-butyl-methyl 6-[(4-fluorophenyl) methyl] -3-methyl-indrin-1,3-dicarboxylate (1.21 g, 3.03 mmol, equivalent: 1) of the racemic form was chirally separated. , (S) -1-tert-Butyl 3-methyl 6- (4-fluorobenzyl) -3-methylindrin-1,3-dicarboxylate (555 mg, 45.9%) was obtained as a colorless oil. MS: m / z M + = 399.2

（Ｓ）−１−ｔｅｒｔ−ブチル ３−メチル ６−（４−フルオロベンジル）−３−メチルインドリン−１，３−ジカルボキシレートを使用して実施例１ｃ）と同じ方法で生成物を調製し、メチル（３Ｓ）−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボキシレート（３６５ｍｇ、９５．５％）を明黄色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝３００．１ b) Methyl (3S) -6-[(4-fluorophenyl) methyl] -3-methyl-indoline-3-carboxylate

(S) Prepare the product in the same manner as in Example 1c) using -1-tert-butyl 3-methyl 6- (4-fluorobenzyl) -3-methylindolin-1,3-dicarboxylate. , Methyl (3S) -6-[(4-fluorophenyl) methyl] -3-methyl-indoline-3-carboxylate (365 mg, 95.5%) was obtained as a bright yellow solid. MS: m / z (M + H) ⁺ = 300.1

（Ｓ）−メチル ６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキシレート（３０３ｍｇ、１．０１ｍｍｏｌ、当量：１）を使用して、実施例１ｄ）と同じ方法で生成物を調製し、メチル（３Ｓ）−１−［２−［（２Ｓ，５Ｒ）−４−ｔｅｒｔ−ブトキシカルボニル−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボキシレート（４４５ｍｇ、６７．３％）を明黄色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６５３．４ c) Methyl (3S) -1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazine] -1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carboxylate

(S) -Methyl 6- (4-fluorobenzyl) -3-methylindrin-3-carboxylate (303 mg, 1.01 mmol, equivalent: 1) was used to prepare the product in the same manner as in Example 1d). Prepared and methyl (3S) -1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl]]. Piperazin-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carboxylate (445 mg, 67.3%) was obtained as a bright yellow oil. MS: m / z (M + H) ⁺ = 653.4

（Ｓ）−メチル １−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキシレート（４４０ｍｇ、６７４μｍｏｌ、当量：１）を使用して、実施例１ｅ）と同じ方法で生成物を調製し、（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（４２０ｍｇ、９７．６％）を白色の泡として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６３９．４ d) (3S) -1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazine- 1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carboxylic acid

(S) -Methyl 1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) ) Acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylate (440 mg, 674 μmol, equivalent: 1) was used to prepare the product in the same manner as in Example 1e). (S) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) Acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylic acid (420 mg, 97.6%) was obtained as white foam. MS: m / z (M + H) ⁺ = 639.4

（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（４１０ｍｇ、６４２μｍｏｌ、当量：１）を使用して、実施例２ａ）と同じ方法で生成物を調製し、（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−６−（４−フルオロベンジル）−３−（（２−メトキシ−２−オキソエチル）カルバモイル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（５０８ｍｇ、１１１％）を明黄色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）＋＝７１０．４ e) (2R, 5S) -tert-butyl 4-(2-((S) -6- (4-fluorobenzyl) -3-((2-methoxy-2-oxoethyl) carbamoyl) -3-methylindoline- 1-yl) -2-oxoethyl) -2-methyl-5-((R) -3-methylmorpholino) methyl) piperazine-1-carboxylate

(S) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) Using acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylic acid (410 mg, 642 μmol, equivalent: 1), the product was prepared in the same manner as in Example 2a). 2R, 5S) -tert-butyl 4-(2-((S) -6- (4-fluorobenzyl) -3-((2-methoxy-2-oxoethyl) carbamoyl) -3-methylindrin-1-yl) ) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (508 mg, 111%) was obtained as a bright yellow oil. MS: m / z (M + H) + = 710.4

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−６−（４−フルオロベンジル）−３−（（２−メトキシ−２−オキソエチル）カルバモイル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（５００ｍｇ、７０４μｍｏｌ、当量：１）を使用して、実施例２ｂ）と同じ方法で生成物を調製し、２−（（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（３８９ｍｇ、７９．４％）を明黄色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６９６．４ f) 2-[[(3S) -1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl]] Methyl] piperazine-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carbonyl] amino] acetic acid

(2R, 5S) -tert-butyl 4-(2-((S) -6- (4-fluorobenzyl) -3-((2-methoxy-2-oxoethyl) carbamoyl) -3-methylindrin-1- Il) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (500 mg, 704 μmol, equivalent: 1), Example 2b ), And 2-((S) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R))) -3-Methylmorpholino) Methyl) Piperazin-1-yl) Acetyl) -6- (4-Fluorobenzyl) -3-Methylindrin-3-Carboxamide) Acetic acid (389 mg, 79.4%) as a bright yellow solid Obtained. MS: m / z (M + H) ⁺ = 696.4

ＥｔＯＡｃ（１３７ｍｇ、１２８μｌ、２１６μｍｏｌ、当量：３）中Ｔ３Ｐ ５０％及びＮ−メチルモルホリン（７２．７ｍｇ、７９μｌ、７１９μｍｏｌ、当量：１０）を、酢酸エチル（１ｍｌ）中、２−（（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（５０ｍｇ、７１．９μｍｏｌ、当量：１）及びｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−１−（（Ｓ）−７−アミノ−３−（（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）カルバモイル）−３，４−ジヒドロイソキノリン−２（１Ｈ）−イル）−３，３−ジメチル−１−オキソブタン−２−イル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（４４．５ｍｇ、７１．９μｍｏｌ、当量：１）（国際公開第２０１３／１９２２８６号に記載）の白色の懸濁液に添加した。室温で１５分間撹拌した後、全てを溶解し、明黄色の溶液を一晩撹拌した。飽和ＮａＨＣＯ_３水溶液を添加し、混合物を酢酸エチルで抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗残留物を、溶出剤として酢酸エチル／メタノール（０−１０％メタノール）を使用したフラッシュクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｓ）−３−［［２−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−３，３−ジメチル−ブタノイル］−３−［［（１Ｒ）−テトラリン−１−イル］カルバモイル］−３，４−ジヒドロ−１Ｈ−イソキノリン−７−イル］アミノ］−２−オキソ−エチル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（６４ｍｇ、６８．６％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２９７．７ g) tert-butyl (2R, 5S) -4- [2-[(3S) -3-[[2-[[(3S) -2-[(2S) -2-[[(2S) -2-] [Tert-Butoxycarbonyl (methyl) amino] propanoyl] amino] -3,3-dimethyl-butanoyl] -3-[[(1R) -tetraline-1-yl] carbamoyl] -3,4-dihydro-1H-isoquinoline -7-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl -5-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazin-1-carboxylate

50% T3P in EtOAc (137 mg, 128 μl, 216 μmol, equivalent: 3) and N-methylmorpholine (72.7 mg, 79 μl, 719 μmol, equivalent: 10) in ethyl acetate (1 ml), 2-((S)-. 1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6 -(4-Fluorobenzyl) -3-methylindrin-3-carboxamide) acetic acid (50 mg, 71.9 μmol, equivalent: 1) and tert-butyl ((S) -1-(((S) -1-(((S) -1-(((S) -1-). S) -7-amino-3-(((R) -1,2,3,4-tetrahydronaphthalene-1-yl) carbamoyl) -3,4-dihydroisoquinolin-2 (1H) -yl) -3, 3-Dimethyl-1-oxobutan-2-yl) amino) -1-oxopropan-2-yl) (methyl) carbamate (44.5 mg, 71.9 μmol, equivalent: 1) (International Publication No. 2013/192286) Described) was added to the white suspension. After stirring at room temperature for 15 minutes, everything was dissolved and the bright yellow solution was stirred overnight. _{Aqueous 3} aqueous solutions of saturated NaHCO were added and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude residue was purified by flash chromatography using ethyl acetate / methanol (0-10% methanol) as eluent and tert-butyl (2R, 5S) -4- [2-[(3S) -3-. [[2-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -3,3-dimethyl-butanoyl]] -3-[[(1R) -tetraline-1-yl] carbamoyl] -3,4-dihydro-1H-isoquinolin-7-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-4-) Fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1 -Carboxylate (64 mg, 68.6%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1297.7

ジオキサン中ＨＣｌ ４Ｍ（１．２ｇ、１ｍｌ、４ｍｍｏｌ、当量：８８）を、酢酸エチル（０．５ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−３，３−ジメチルブタノイル）−３−（（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）カルバモイル）−１，２，３，４−テトラヒドロイソキノリン−７−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（５９ｍｇ、４５．５μｍｏｌ、当量：１）の明黄色の溶液に添加し、反応混合物を室温で２時間撹拌した。白色の沈殿物を濾別し、ジエチルエーテルで洗浄し、真空中で乾燥させて、（３Ｓ）−２−［（２Ｓ）−３，３−ジメチル−２−［［（２Ｓ）−２−（メチルアミノ）プロパノイル］アミノ］ブタノイル］−７−［［２−［［（３Ｓ）−６−［（４−フルオロフェニル）メチル］−３−メチル−１−［２−［（２Ｒ，５Ｒ）−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］インドリン−３−カルボニル］アミノ］アセチル］アミノ］−Ｎ−［（１Ｒ）−テトラリン−１−イル］−３，４−ジヒドロ−１Ｈ−イソキノリン−３−カルボキサミド トリヒドロクロリド（４８ｍｇ、８７．５％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０９７．６ h) (3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2- (methylamino) propanoyl] amino] butanoyl] -7-[[2-[[(3S) ) -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholine-4-yl) ] Methyl] piperazin-1-yl] acetyl] indolin-3-carbonyl] amino] acetyl] amino] -N-[(1R) -tetraline-1-yl] -3,4-dihydro-1H-isoquinolin-3-3 Carboxamide trihydrochloride

HCl 4M (1.2 g, 1 ml, 4 mmol, equivalent: 88) in dioxane in (2R, 5S) -tert-butyl 4-(2-((S) -3-(((S) -3-(()) in ethyl acetate (0.5 ml). 2-(((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -3,3-dimethylbutanoyl) -3 -(((R) -1,2,3,4-tetrahydronaphthalene-1-yl) carbamoyl) -1,2,3,4-tetrahydroisoquinolin-7-yl) amino) -2-oxoethyl) carbamoyl)- 6- (4-Fluorobenzyl) -3-methylindolin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate ( It was added to a bright yellow solution of 59 mg, 45.5 μmol, equivalent: 1), and the reaction mixture was stirred at room temperature for 2 hours. The white precipitate was filtered off, washed with diethyl ether, dried in vacuum and (3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2-( Methylamino) propanoyl] amino] butanoyl] -7-[[2-[[(3S) -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R)-] 5-Methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carbonyl] amino] acetyl] amino] -N-[(1R) -Tetraline-1-yl] -3,4-dihydro-1H-isoquinolin-3-carboxamide Trihydrochloride (48 mg, 87.5%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1097.6

ＥｔＯＡｃ（１３７ｍｇ、１２８μｌ、２１６μｍｏｌ、当量：３）中Ｔ３Ｐ ５０％及びＮ−メチルモルホリン（７２．７ｍｇ、７９μｌ、７１９μｍｏｌ、当量：１０）を、酢酸エチル（１ｍｌ）中２−（（Ｒ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（５０ｍｇ、７１．９μｍｏｌ、当量：１）及びｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−１−（（Ｓ）−７−アミノ−３−（（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）カルバモイル）−３，４−ジヒドロイソキノリン−２（１Ｈ）−イル）−３，３−ジメチル−１−オキソブタン−２−イル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（４４．５ｍｇ、７１．９μｍｏｌ、当量：１）の白色の懸濁液に添加した。室温で１５分間撹拌した後、全てを溶解し、明黄色の溶液を一晩撹拌した。飽和ＮａＨＣＯ_３水溶液を添加し、混合物を酢酸エチルで抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗残留物を、溶出剤として酢酸エチル／メタノール（０−１０％メタノール）を使用したフラッシュクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｒ）−３−［［２−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−３，３−ジメチル−ブタノイル］−３−［［（１Ｒ）−テトラリン−１−イル］カルバモイル］−３，４−ジヒドロ−１Ｈ−イソキノリン−７−イル］アミノ］−２−オキソ−エチル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（５４ｍｇ、５７．９％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２９７．７ Example 11
(3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2- (methylamino) propanoyl] amino] butanoyl] -7-[[2-[[(3R)- 6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] ] Piperazin-1-yl] acetyl] indolin-3-carbonyl] amino] acetyl] amino] -N-[(1R) -1,2,3,4-tetrahydronaphthalene-1-yl] -3,4-dihydro -1H-isoquinolin-3-carboxamide trihydrochloride a) tert-butyl (2R, 5S) -4- [2-[(3R) -3-[[2-[[(3S) -2-[(2S)) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -3,3-dimethyl-butanoyl] -3-[[(1R) -tetraline-1-yl] carbamoyl ] -3,4-dihydro-1H-isoquinoline-7-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate

50% T3P in EtOAc (137 mg, 128 μl, 216 μmol, equivalent: 3) and N-methylmorpholine (72.7 mg, 79 μl, 719 μmol, equivalent: 10) in 2-((R) -1) in ethyl acetate (1 ml). -(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6- (4-Fluorobenzyl) -3-methylindrin-3-carboxamide) Acetic acid (50 mg, 71.9 μmol, equivalent: 1) and tert-butyl ((S) -1-(((S) -1-((S) -1-((S) ) -7-Amino-3-(((R) -1,2,3,4-tetrahydronaphthalene-1-yl) carbamoyl) -3,4-dihydroisoquinolin-2 (1H) -yl) -3,3 -Dimethyl-1-oxobutan-2-yl) amino) -1-oxopropan-2-yl) (methyl) carbamate (44.5 mg, 71.9 μmol, equivalent: 1) was added to a white suspension. After stirring at room temperature for 15 minutes, everything was dissolved and the bright yellow solution was stirred overnight. _{Aqueous 3} aqueous solutions of saturated NaHCO were added and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude residue was purified by flash chromatography using ethyl acetate / methanol (0-10% methanol) as eluent and tert-butyl (2R, 5S) -4- [2-[(3R) -3-. [[2-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -3,3-dimethyl-butanoyl]] -3-[[(1R) -tetraline-1-yl] carbamoyl] -3,4-dihydro-1H-isoquinolin-7-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-4-) Fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1 -Carboxylate (54 mg, 57.9%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1297.7

ジオキサン中ＨＣｌ ４Ｍ（９６３μｌ、３．８５ｍｍｏｌ、当量：１００）を、酢酸エチル（０．５ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｒ）−３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−３，３−ジメチルブタノイル）−３−（（（Ｒ）−１，２，３，４−テトラヒドロナフタレン−１−イル）カルバモイル）−１，２，３，４−テトラヒドロイソキノリン−７−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（５０ｍｇ、３８．５μｍｏｌ、当量：１）の明黄色の溶液に添加し、反応混合物を室温で２時間撹拌した。白色の沈殿物を濾別し、ジエチルエーテルで洗浄し、真空中で乾燥させて、（３Ｓ）−２−［（２Ｓ）−３，３−ジメチル−２−［［（２Ｓ）−２−（メチルアミノ）プロパノイル］アミノ］ブタノイル］−７−［［２−［［（３Ｒ）−６−［（４−フルオロフェニル）メチル］−３−メチル−１−［２−［（２Ｒ，５Ｒ）−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］インドリン−３−カルボニル］アミノ］アセチル］アミノ］−Ｎ−［（１Ｒ）−１，２，３，４−テトラヒドロナフタレン −１−イル］−３，４−ジヒドロ−１Ｈ−イソキノリン−３−カルボキサミド トリヒドロクロリド（４０ｍｇ、８６％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０９７．６ b) (3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2- (methylamino) propanoyl] amino] butanoyl] -7-[[2-[[(3R) ) -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholine-4-yl) ] Methyl] piperazin-1-yl] acetyl] indolin-3-carbonyl] amino] acetyl] amino] -N-[(1R) -tetraline-1-yl] -3,4-dihydro-1H-isoquinolin-3-3 Carboxamide trihydrochloride

HCl 4M (963 μl, 3.85 mmol, equivalent: 100) in dioxane in (2R, 5S) -tert-butyl 4-(2-((R) -3-((2-)) in ethyl acetate (0.5 ml). (((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -3,3-dimethylbutanoyl) -3-( ((R) -1,2,3,4-tetrahydronaphthalene-1-yl) carbamoyl) -1,2,3,4-tetrahydroisoquinolin-7-yl) amino) -2-oxoethyl) carbamoyl) -6- (4-Fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (50 mg, It was added to a bright yellow solution of 38.5 μmol, equivalent: 1), and the reaction mixture was stirred at room temperature for 2 hours. The white precipitate was filtered off, washed with diethyl ether, dried in vacuo and (3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2-( Methylamino) propanoyl] amino] butanoyl] -7-[[2-[[(3R) -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R)-) 5-Methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carbonyl] amino] acetyl] amino] -N-[(1R) -1,2,3,4-tetrahydronaphthalene-1-yl] -3,4-dihydro-1H-isoquinoline-3-carboxamide trihydrochloride (40 mg, 86%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1097.6

（Ｒ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（８０ｍｇ、１２５μｍｏｌ、当量：１）を、酢酸エチル（４８０μｌ）中でｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（７７．１ｍｇ、１２５μｍｏｌ、当量：１；中間体２）、Ｎ−メチルモルホリン（１２７ｍｇ、１３８μｌ、１．２５ｍｍｏｌ、当量：１０）及びＥｔＯＡｃ ５０％（２３９ｍｇ、２２４μｌ、３７６μｍｏｌ、当量：３）中Ｔ３Ｐと室温で週末にわたって撹拌した。飽和ＮａＨＣＯ３溶液を添加し、混合物を酢酸エチルで３回抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチルを使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｒ）−３−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（９６ｍｇ、６２％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２３６．６ Example 12
(R) -N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl) -2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride
a) tert-Butyl (2R, 5S) -4- [2-[(3R) -3-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxy] Carbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] carbamoyl] -6-[((2,6-difluorophenyl) carbamoyl] 4-Fluorophenyl) Methyl] -3-Methyl-Indoline-1-yl] -2-oxo-Ethyl] -2-Methyl-5-[[(3R) -3-Methylmorpholin-4-yl] Methyl] Piperazine -1-carboxylate

(R) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) Acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylic acid (80 mg, 125 μmol, equivalent: 1) was added to tert-butyl ((S) -1- ((S) -1-) in ethyl acetate (480 μl). ((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindolin-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-) 4-Il) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (77.1 mg, 125 μmol, equivalent: 1; intermediate 2), N-methylmorpholin (127 mg, 138 μl, 1.25 mmol) , Equivalent: 10) and EtOAc in 50% (239 mg, 224 μl, 376 μmol, equivalent: 3) with T3P and stirred at room temperature over the weekend. A saturated NaHCO3 solution was added and the mixture was extracted 3 times with ethyl acetate. The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude substance was applied to silica gel, purified by column chromatography using ethyl acetate as an eluent, and tert-butyl (2R, 5S) -4- [2-[(3R) -3-[[(3S)-). 2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2, 6-Difluorophenyl) carbamoyl] isoindrin-5-yl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl -5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (96 mg, 62%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1236.6

ジオキサン（１．８２ｍｌ、７．２８ｍｍｏｌ、当量：１００）中ＨＣｌ ４Ｍを、酢酸エチル（１ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｒ）−３−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（９０ｍｇ、７２．８μｍｏｌ、当量：１）の透明な溶液に添加し、反応混合物を室温で２時間撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄し、乾燥させて、（Ｒ）−Ｎ−（（Ｓ）−３−（（２，６−ジフルオロフェニル）カルバモイル）−２−（（Ｓ）−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−５−イル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド トリヒドロクロリド （７８ｍｇ、９３．５％）を オフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０３６．５ b) (3R) -N-[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino) propanoyl]] Amino] -2-tetrahydropyran-4-yl-acetyl] isoindoline-5-yl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R)- 5-Methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indoline-3-carboxamide trihydrochloride

HCl 4M in dioxane (1.82 ml, 7.28 mmol, equivalent: 100), (2R, 5S) -tert-butyl 4-(2-((R) -3-(((S)) in ethyl acetate (1 ml)). ) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl)- 3-((2,6-difluorophenyl) carbamoyl) isoindolin-5-yl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindolin-1-yl) -2-oxoethyl) -2-methyl It was added to a clear solution of −5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (90 mg, 72.8 μmol, equivalent: 1) and the reaction mixture was stirred at room temperature for 2 hours. .. The precipitate was filtered off, washed with diethyl ether, dried and (R) -N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2). -((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-5-yl) -6- (4-fluorobenzyl) -3- Methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indoline-3-carboxamide trihydrochloride ( 78 mg, 93.5%) was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 1036.5

Ｎ−メチルモルホリン（１１６ｍｇ、１２６μｌ、１．１５ｍｍｏｌ、当量：１０）及びＥｔＯＡｃ中Ｔ３Ｐ ５０％（２１９ｍｇ、２０５μｌ、３４５μｍｏｌ、当量：３）を、酢酸エチル（１．５ｍｌ）中２−（１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（８０ｍｇ、１１５μｍｏｌ、当量：１）及びｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−７−アミノ−３−（（２，６−ジフルオロフェニル）カルバモイル）−３，４−ジヒドロイソキノリン−２（１Ｈ）−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（７２．４ｍｇ、１１５μｍｏｌ、当量：１；中間体３）の白色の懸濁液に添加した。室温で１５分間撹拌した後、全てを溶解し、明黄色の溶液を一晩撹拌した。飽和ＮａＨＣＯ３水溶液を添加し、混合物を酢酸エチルで抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗残留物を、溶出剤として酢酸エチル／メタノール（０−１０％メタノール）を使用したフラッシュクロマトグラフィーにより精製し、（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）−１，２，３，４−テトラヒドロイソキノリン−７−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（２９ｍｇ、１９．３％）をオフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１３０７．７ Example 13
a) (3S) -N- (2,6-difluorophenyl) -7- (2- (6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-) Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) acetamide) -2-((S) -2-((S) -2-) (Methylamino) Propanamide) -2- (Tetrahydro-2H-Pyran-4-yl) Acetyl) -1,2,3,4-Tetrahydroisoquinolin-3-Carboxamide Trihydrochloride tert-butyl (2R, 5S)- 4- [2- [3-[[2-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino]- 2-Tetrahydropyran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] -3,4-dihydro-1H-isoquinoline-7-yl] amino] -2-oxo-ethyl] carbamoyl ] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4 -Il] Methyl] Piperazin-1-carboxylate

N-Methylmorpholin (116 mg, 126 μl, 1.15 mmol, equivalent: 10) and 50% T3P in EtOAc (219 mg, 205 μl, 345 μmol, equivalent: 3), 2- (1- (1- (1.5 ml)) in ethyl acetate. 2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6- (4) -Fluorobenzyl) -3-methylindrin-3-carboxamide) Acetic acid (80 mg, 115 μmol, equivalent: 1) and tert-butyl ((S) -1-(((S) -2-((S) -7-) Amino-3-((2,6-difluorophenyl) carbamoyl) -3,4-dihydroisoquinolin-2 (1H) -yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) Amino) -1-oxopropan-2-yl) (methyl) carbamate (72.4 mg, 115 μmol, equivalent: 1; intermediate 3) was added to a white suspension. After stirring at room temperature for 15 minutes, everything was dissolved and the bright yellow solution was stirred overnight. A saturated aqueous NaHCO3 solution was added and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude residue was purified by flash chromatography using ethyl acetate / methanol (0-10% methanol) as eluent and (2R, 5S) -tert-butyl 4- (2- (3-((2-)). (((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) ) Acetyl) -3-((2,6-difluorophenyl) carbamoyl) -1,2,3,4-tetrahydroisoquinolin-7-yl) amino) -2-oxoethyl) carbamoyl) -6- (4-fluorobenzyl) ) -3-Methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (29 mg, 19.3%) Was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 1307.7

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）−１，２，３，４−テトラヒドロイソキノリン−７−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（２５ｍｇ、１９．１μｍｏｌ、当量：１）を、酢酸エチル（６００μＬ）中でジオキサン ４Ｍ（４００μｌ、１．６ｍｍｏｌ、当量：８３．７）中ＨＣｌと室温で一晩撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄し、高減圧下で乾燥させて、（３Ｓ）−Ｎ−（２，６−ジフルオロフェニル）−７−（２−（６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド）アセトｔアミド）−２−（（Ｓ）−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−１，２，３，４−テトラヒドロイソキノリン−３−カルボキサミド トリヒドロクロリド （１４ｍｇ、６０．２％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１１０７．６ b) (3S) -N- (2,6-difluorophenyl) -7-[[2-[[6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R,) 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carbonyl] amino] acetyl] amino] -2- [ (2S) -2-[[(2S) -2- (methylamino) propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3,4-dihydro-1H-isoquinolin-3-carboxamide trihydro Chloride

(2R, 5S) -tert-butyl 4-(2-(3-((2-(((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl)) (Methyl) Amino) Propanamide) -2- (Tetrahydro-2H-Pyran-4-yl) Acetyl) -3-((2,6-Difluorophenyl) Carbamoyl) -1,2,3,4-Tetrahydroisoquinolin- 7-Il) Amino) -2-oxoethyl) Carbamoyl) -6- (4-Fluorobenzyl) -3-Methylindrin-1-yl) -2-oxoethyl) -2-Methyl-5-(((R)-)- 3-Methylmorpholino) Methyl) piperazin-1-carboxylate (25 mg, 19.1 μmol, equivalent: 1) in ethyl acetate (600 μL) in 4M (400 μl, 1.6 mmol, equivalent: 83.7) HCl And stirred overnight at room temperature. The precipitate was filtered off, washed with diethyl ether, dried under high vacuum and (3S) -N- (2,6-difluorophenyl) -7- (2- (6- (4-fluorobenzyl)). -3-Methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) Acet tamide) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) -1,2,3 , 4-Tetrahydroisoquinolin-3-carboxamide trihydrochloride (14 mg, 60.2%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1107.6

（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボン酸（５００ｍｇ、１．０２ｍｍｏｌ、当量：１；中間体１）、ベンジル ３−アミノ−４−フルオロベンゾエート（２７６ｍｇ、１．１２ｍｍｏｌ、当量：１．１）及びＥｔＯＡｃ（１．３ｇ、１．２２ｍｌ、２．０４ｍｍｏｌ、当量：２）中Ｔ３Ｐ ５０％を酢酸エチル（３．５ｍｌ）中に溶解した。ピリジン（１．７５ｍｌ）を添加し、黄色の溶液を室温で一晩撹拌した。反応混合物を酢酸エチルに注ぎ、希釈ＨＣｌ及びブラインで抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質を、溶出剤として酢酸エチル／ヘプタン（０−１００％酢酸エチル）を使用したフラッシュクロマトグラフィーにより精製し、ベンジル ３−（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキサミド）−４−フルオロベンゾエート（５１０ｍｇ、６９．７％）を白色の泡として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝７１７．３３ Example 14
N- (2- (4-Fluoro-3-((S) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-) 4-yl) Acetyl) Isoindrin-1-Carboxamide) Benzamide) Ethyl) -6- (4-Fluorobenzyl) -3-Methyl-1-(2-((2R, 5R) -5-Methyl-2- ( ((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide dihydrochloride a) benzyl 3-[[(1S) -2-[(2S) -2-[[( 2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-1-carbonyl] amino] -4-fluoro-benzoate

(S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl ) Isoindrin-1-carboxylic acid (500 mg, 1.02 mmol, equivalent: 1; intermediate 1), benzyl 3-amino-4-fluorobenzoate (276 mg, 1.12 mmol, equivalent: 1.1) and EtOAc (1) .3 g, 1.22 ml, 2.04 mmol, equivalent: 2) 50% of T3P was dissolved in ethyl acetate (3.5 ml). Pyridine (1.75 ml) was added and the yellow solution was stirred at room temperature overnight. The reaction mixture was poured into ethyl acetate and extracted with diluted HCl and brine. The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude material was purified by flash chromatography using ethyl acetate / heptane (0-100% ethyl acetate) as an eluent and benzyl 3-((S) -2-((S) -2-((S)). -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-1-carboxamide) -4-fluorobenzoate (510 mg, 69) .7%) was obtained as white foam. MS: m / z (M + H) ⁺ = 717.33

Ｐｄ−Ｃ １０％（６５ｍｇ、６１．１μｍｏｌ、当量：０．０９７３）を、メタノール（１５ｍｌ）中ベンジル ３−（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキサミド）−４−フルオロベンゾエート（４５０ｍｇ、６２８μｍｏｌ、当量：１）の透明な溶液に添加し、反応物を水素雰囲気下で一晩撹拌した。反応混合物をデカライトで濾過し、濃縮して、３−（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキサミド）−４−フルオロ安息香酸（３７５ｍｇ、９５．３％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６２７．２８ b) 3-[[(1S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl -Acetyl] isoindoline-1-carbonyl] amino] -4-fluoro-benzoic acid

Pd-C 10% (65 mg, 61.1 μmol, equivalent: 0.0973) in benzyl 3-((S) -2-((S) -2-((S) -2-() in methanol (15 ml)) (Tart-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-1-carboxamide) -4-fluorobenzoate (450 mg, 628 μmol, equivalent: 1) ) Was added to the clear solution, and the reaction was stirred overnight under a hydrogen atmosphere. The reaction mixture is filtered through decalite and concentrated to 3-((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide)). -2- (Tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-1-carboxamide) -4-fluorobenzoic acid (375 mg, 95.3%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 627.28

３−（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキサミド）−４−フルオロ安息香酸（１２０ｍｇ、１９１μｍｏｌ、当量：１）を、ベンジル（２−アミノエチル）カルバメート ヒドロクロリド（４４．２ｍｇ、１９１μｍｏｌ、当量：１）、ＤＩＰＥＡ（９９ｍｇ、１３４μｌ、７６６μｍｏｌ、当量：４）及びＤＭＦ（１．７ｍｌ）中ＨＡＴＵ（９４．７ｍｇ、２４９μｍｏｌ、当量：１．３）と室温で一晩撹拌した。０．５Ｍ ＨＣｌを添加し、酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲル上に適用し、溶出剤として酢酸エチル／メタノール（０−５％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−１−（（５−（（２−（（（ベンジルオキシ）カルボニル）アミノ）エチル）カルバモイル）−２−フルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（１５３ｍｇ、９９．５％）を無色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝８０３．３８ c) tert-Butyl N-[(1S) -2-[[(1S) -2-[(1S) -1-[[5- [2- (benzyloxycarbonylamino) ethylcarbamoyl] -2-fluoro- Phenyl] carbamoyl] isoindoline-2-yl] -2-oxo-1-tetrahydropyran-4-yl-ethyl] amino] -1-methyl-2-oxo-ethyl] -N-methyl-carbamate

3-((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-) Il) Acetyl) Isoindrin-1-carboxamide) -4-fluorobenzoic acid (120 mg, 191 μmol, equivalent: 1), benzyl (2-aminoethyl) carbamate hydrochloride (44.2 mg, 191 μmol, equivalent: 1), It was stirred overnight at room temperature with HATU (94.7 mg, 249 μmol, eq: 1.3) in DIPEA (99 mg, 134 μl, 766 μmol, eq: 4) and DMF (1.7 ml). 0.5 M HCl was added, extracted 3 times with ethyl acetate, dried over magnesium sulphate, filtered and evaporated. The crude substance was applied on silica gel and purified by column chromatography using ethyl acetate / methanol (0-5% methanol) as an eluent, and tert-butyl ((S) -1-(((S) -2) -((S) -1-((5-((2-(((benzyloxy) carbonyl) amino) ethyl) carbamoyl) -2-fluorophenyl) carbamoyl) isoindolin-2-yl) -2-oxo- 1- (Tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (153 mg, 99.5%) was obtained as a colorless oil. MS: m / z (M + H) ⁺ = 803.38

ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−１−（（５−（（２−（（（ベンジルオキシ）カルボニル）アミノ）エチル）カルバモイル）−２−フルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（１２０ｍｇ、１４９μｍｏｌ、当量：１）を、メタノール（５ｍｌ）中パラジウム炭素１０％（１６ｍｇ、１５μｍｏｌ、当量：０．１０１）と水素雰囲気下室温で一晩撹拌した。触媒を濾別し、溶媒をエバポレートして、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−１−（（５−（（２−アミノエチル）カルバモイル）−２−フルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（９０ｍｇ、９０％）を明黄色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６６９．３４ d) tert-Butyl N-[(1S) -2-[[(1S) -2-[(1S) -1-[[5- (2-aminoethylcarbamoyl) -2-fluoro-phenyl] carbamoyl] iso Indoline-2-yl] -2-oxo-1-tetrahydropyran-4-yl-ethyl] amino] -1-methyl-2-oxo-ethyl] -N-methyl-carbamate

tert-butyl ((S) -1-(((S) -2-((S) -1-((5-((2-(((benzyloxy) carbonyl) amino) ethyl) carbamoyl) -2- Fluorophenyl) carbamoyl) isoindolin-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (120 mg) 149 μmol, equivalent: 1) was stirred overnight at room temperature under a hydrogen atmosphere with 10% (16 mg, 15 μmol, equivalent: 0.101) of palladium carbon in methanol (5 ml). The catalyst was filtered off and the solvent was evaporated to tert-butyl ((S) -1-(((S) -2-((S) -1-((5-((2-aminoethyl) carbamoyl)). -2-Fluorophenyl) Carbamoyl) Isoindoline-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) Amino) -1-oxopropan-2-yl) (methyl) Carbamate (90 mg, 90%) was obtained as a bright yellow oil. MS: m / z (M + H) ⁺ = 669.34

ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−１−（（５−（（２−アミノエチル）カルバモイル）−２−フルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（８５ｍｇ、１２７μｍｏｌ、当量：１）を、ＤＩＰＥＡ（６５．７ｍｇ、８８．８μｌ、５０８μｍｏｌ、当量：４）及びＤＭＦ（１ｍｌ）中ＨＡＴＵ（６２．８ｍｇ、１６５μｍｏｌ、当量：１．３）の存在下、１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（８１．２ｍｇ、１２７μｍｏｌ、当量：１）と室温で一晩撹拌した。飽和ＮａＨＣＯ_３ ｓｏｌ． ａｑ．を添加し、硫酸エチルで３回抽出し、硫酸ナトリウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−５％メタノール）を使用したカラムクロマトグラフィーにより精製し、（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（２−（３−（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキサミド）−４−フルオロベンズアミド）エチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（３１ｍｇ、１８．９％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２８９．６８ e) tert-butyl (2R, 5S) -4- [2- [3- [2-[[3-[[(1S) -2-[(2S) -2-[[(2S) -2-[ tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-1-carbonyl] amino] -4-fluoro-benzoyl] amino] ethylcarbamoyl] -6- [ (4-Fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] Piperazin-1-carboxylate

tert-butyl ((S) -1-(((S) -2-((S) -1-((5-((2-aminoethyl) carbamoyl) -2-fluorophenyl) carbamoyl) isoindrin-2) -Il) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (85 mg, 127 μmol, equivalent: 1), 1- (2-((2S, 5R) ) -4- (tert-Butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6- (4-fluorobenzyl) -3-3 Methylindrin-3-carboxylic acid (81.2 mg, 127 μmol, equivalent: 1) was stirred overnight at room temperature. Saturated NaHCO ₃ sol. aq. Was added, extracted 3 times with ethyl sulfate, dried over sodium sulfate, filtered and evaporated. The crude material was applied to silica gel and purified by column chromatography using ethyl acetate / methanol (0-5% methanol) as eluent and purified by (2R, 5S) -tert-butyl 4- (2- (3- (3- (3). (2-(3-((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-) Piran-4-yl) Acetyl) Isoindrin-1-Carboxamide) -4-Fluorobenzamide) Ethyl) Carbamoyl) -6- (4-Fluorobenzyl) -3-Methylindrin-1-yl) -2-oxoethyl)- 2-Methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (31 mg, 18.9%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1289.68

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（２−（３−（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキサミド）−４−フルオロベンズアミド）エチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（３０ｍｇ、２３．３μｍｏｌ、当量：１）を、酢酸エチル（５００μＬ）中で、ジオキサン４Ｍ（５００μｌ、２ｍｍｏｌ、当量：８６）中ＨＣｌと室温で週末にわたって撹拌し、沈殿した固体を濾別し、ジエチルエーテルで洗浄し、高減圧下で乾燥させて、Ｎ−［２−［［４−フルオロ−３−［［（１Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−（メチルアミノ）プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］イソインドリン−１−カルボニル］アミノ］ベンゾイル］アミノ］エチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−１−［２−［（２Ｒ，５Ｒ）−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］インドリン−３−カルボキサミド ジヒドロクロリド（２３．１６ｍｇ、８５．７％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０８９．５８ f) N- [2-[[4-Fluor-3-[[(1S) -2-[(2S) -2-[[(2S) -2- (methylamino) propanoyl] amino] -2-tetrahydro Piran-4-yl-acetyl] isoindolin-1-carbonyl] amino] benzoyl] amino] ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) ) -5-Methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide dihydrochloride

(2R, 5S) -tert-butyl 4-(2-(3-((2-(3-((S) -2-((S) -2-((S) -2-((tert-butoxy) Carbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-1-carboxamide) -4-fluorobenzamide) ethyl) carbamoyl) -6- (4-fluoro) Benzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (30 mg, 23.3 μmol, Equivalent: 1) was stirred in ethyl acetate (500 μL) with HCl in Dioxane 4M (500 μl, 2 mmol, equivalent: 86) over the weekend at room temperature, the precipitated solid was filtered off, washed with diethyl ether and high. Dry under reduced pressure and N- [2-[[4-fluoro-3-[[(1S) -2-[(2S) -2-[[(2S) -2- (methylamino) propanoyl] amino] ] -2-Tetrahydropyran-4-yl-acetyl] isoindolin-1-carbonyl] amino] benzoyl] amino] ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-] [(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide dihydrochloride (23.16 mg, 85.7%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1089.58

（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（８０ｍｇ、１２５μｍｏｌ、当量：１）を、酢酸エチル（５００μｌ）中でｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（７７．１ｍｇ、１２５μｍｏｌ、当量：１；中間体２）、Ｎ−メチルモルホリン（１２７ｍｇ、１３８μｌ、１．２５ｍｍｏｌ、当量：１０）及びＥｔＯＡｃ ５０％（２３９ｍｇ、２２４μｌ、３７６μｍｏｌ、当量：３）中Ｔ３Ｐと室温で週末にわたって撹拌した。飽和ＮａＨＣＯ３溶液を添加し、混合物を酢酸エチルで３回抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチルを使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｓ）−３−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（７６ｍｇ、４９．１％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２３６．６ Example 15
a) (S) -N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide)) -2- (Tetrahydro-2H-pyran-4-yl) acetyl) Isoindrin-5-yl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5) -Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide tert-butyl (2R, 5S) -4- [2-[2-[(3S)- 3-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl ] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indrin-1-yl] -2- Oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate

(S) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) Acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylic acid (80 mg, 125 μmol, equivalent: 1) was added to tert-butyl ((S) -1- ((S) -1-) in ethyl acetate (500 μl). ((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindolin-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-) 4-Il) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (77.1 mg, 125 μmol, equivalent: 1; intermediate 2), N-methylmorpholin (127 mg, 138 μl, 1.25 mmol) , Equivalent: 10) and EtOAc in 50% (239 mg, 224 μl, 376 μmol, equivalent: 3) with T3P and stirred at room temperature over the weekend. A saturated NaHCO3 solution was added and the mixture was extracted 3 times with ethyl acetate. The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude substance was applied to silica gel, purified by column chromatography using ethyl acetate as an eluent, and tert-butyl (2R, 5S) -4- [2-[(3S) -3-[[(3S)-). 2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2, 6-Difluorophenyl) carbamoyl] isoindrin-5-yl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl -5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (76 mg, 49.1%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1236.6

ジオキサン（１．３７ｍｌ、５．５ｍｍｏｌ、当量：１００）中ＨＣｌ ４Ｍを、酢酸エチル（０．５ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（６８ｍｇ、５５μｍｏｌ、当量：１）の明黄色の溶液に添加し、反応物を室温で２時間撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄し、乾燥させた。物質を分取ＨＰＬＣにより精製し、（Ｓ）−Ｎ−（（Ｓ）−３−（（２，６−ジフルオロフェニル）カルバモイル）−２−（（Ｓ）−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−５−イル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド（２３ｍｇ、４０．４％）を明黄色の非晶質として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０３６．５ b) (3S) -N-[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino) propanoyl]] Amino] -2-tetrahydropyran-4-yl-acetyl] isoindoline-5-yl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R)- 5-Methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indoline-3-carboxamide

HCl 4M in dioxane (1.37 ml, 5.5 mmol, equivalent: 100) and (2R, 5S) -tert-butyl 4-(2-((S) -3-(((S) -3-(()) in ethyl acetate (0.5 ml). (S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl ) -3-((2,6-difluorophenyl) carbamoyl) isoindoline-5-yl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2 -Methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (68 mg, 55 μmol, equivalent: 1) was added to a bright yellow solution and the reaction was stirred at room temperature for 2 hours. bottom. The precipitate was filtered off, washed with diethyl ether and dried. The substance was purified by preparative HPLC and (S) -N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2-) (Methylamino) Propanamide) -2- (Tetrahydro-2H-Pyran-4-yl) Acetyl) Isoindoline-5-yl) -6- (4-Fluorobenzyl) -3-Methyl-1- (2- (2- ( (2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indoline-3-carboxamide (23 mg, 40.4%) in bright yellow Obtained as amorphous. MS: m / z (M + H) ⁺ = 1036.5

３−（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキサミド）−４−フルオロ安息香酸（１２０ｍｇ、１９１μｍｏｌ、当量：１）を、酢酸エチル（２．５ｍｌ）中でベンジル（４−アミノフェニル）カルバメート（５３．４ｍｇ、２２０μｍｏｌ、当量：１．１５）、Ｎ−メチルモルホリン（１９４ｍｇ、２１１μｌ、１．９１ｍｍｏｌ、当量：１０）及びＥｔＯＡｃ（３６６ｍｇ、３４２μｌ、５７４μｍｏｌ、当量：３）中Ｔ３Ｐ ５０％と室温で一晩撹拌した。飽和ＮａＨＣＯ_３ａｑ．を添加し、硫酸エチルで３回抽出し、硫酸ナトリウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−５％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−１−（（５−（（４−（（（ベンジルオキシ）カルボニル）アミノ）フェニル）カルバモイル）−２−フルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（１０３ｍｇ、６３．２％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ−Ｈ）⁻＝８４９．３６ Example 16
N- (4- (4-Fluoro-3-((S) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-) 4-Il) Acetyl) Isoindrin-1-Carboxamide) Benzamide) Phenyl) -6- (4-Fluorobenzyl) -3-Methyl-1-(2-((2R, 5R) -5-Methyl-2- ( ((R) -3-Methylmorpholino) Methyl) Piperazin-1-yl) Acetyl) Indolin-3-Carboxamide Trihydrochloride a) tert-butyl N-[(1S) -2-[[(1S) -2-] [(1S) -1-[[5-[[4- (benzyloxycarbonylamino) phenyl] carbamoyl] -2-fluoro-phenyl] carbamoyl] isoindolin-2-yl] -2-oxo-1-tetrahydropyran -4-Il-ethyl] amino] -1-methyl-2-oxo-ethyl] -N-methyl-carbamate

3-((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-) Il) Acetyl) Isoindrin-1-carboxamide) -4-fluorobenzoic acid (120 mg, 191 μmol, equivalent: 1) in ethyl acetate (2.5 ml) with benzyl (4-aminophenyl) carbamate (53.4 mg,). Stirred overnight at room temperature with 50% T3P in 220 μmol, equivalent: 1.15), N-methylmorpholine (194 mg, 211 μl, 1.91 mmol, equivalent: 10) and EtOAc (366 mg, 342 μl, 574 μmol, equivalent: 3). .. Saturated NaHCO ₃ aq. Was added, extracted 3 times with ethyl sulfate, dried over sodium sulfate, filtered and evaporated. The crude substance was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-5% methanol) as an eluent, and tert-butyl ((S) -1-(((S) -2-)). ((S) -1-((5-((4-(((benzyloxy) carbonyl) amino) phenyl) carbamoyl) -2-fluorophenyl) carbamoyl) isoindolin-2-yl) -2-oxo-1 -(Tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (103 mg, 63.2%) was obtained as a white solid. MS: m / z (MH) ^- = 849.36

ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−１−（（５−（（４−（（（ベンジルオキシ）カルボニル）アミノ）フェニル）カルバモイル）−２−フルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（９７ｍｇ、１１４μｍｏｌ、当量：１）を、メタノール（２．８ｍｌ）中でＰｄ−Ｃ １０％（１５ｍｇ、１４．１μｍｏｌ、当量：０．１２４）と水素雰囲気下室温で週末にわたって撹拌した。触媒を濾別し、溶媒をエバポレートし、高減圧下で乾燥させて、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−１−（（５−（（４−アミノフェニル）カルバモイル）−２−フルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（８２ｍｇ、１００ ％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝７１７．３４ b) tert-Butyl N-[(1S) -2-[[(1S) -2-[(1S) -1-[[5-[(4-aminophenyl) carbamoyl] -2-fluoro-phenyl] carbamoyl] ] Isoindoline-2-yl] -2-oxo-1-tetrahydropyran-4-yl-ethyl] amino] -1-methyl-2-oxo-ethyl] -N-methyl-carbamate

tert-butyl ((S) -1-(((S) -2-((S) -1-((5-((4-(((benzyloxy) carbonyl) amino) phenyl) carbamoyl) -2- Fluorophenyl) carbamoyl) isoindoline-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (97 mg) , 114 μmol, equivalent: 1) was stirred in methanol (2.8 ml) with 10% Pd-C (15 mg, 14.1 μmol, equivalent: 0.124) over a weekend at room temperature in a hydrogen atmosphere. The catalyst is filtered off, the solvent is evaporated and dried under high reduced pressure to allow tert-butyl ((S) -1-(((S) -2-((S) -1-((5-(((5-((). 4-aminophenyl) carbamoyl) -2-fluorophenyl) carbamoyl) isoindoline-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropane- 2-Il) (methyl) carbamate (82 mg, 100%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 717.34

ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−１−（（５−（（４−アミノフェニル）カルバモイル）−２−フルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（７８ｍｇ、１０９μｍｏｌ、当量：１）を、酢酸エチル（１．５ｍｌ）中で１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（７６．５ｍｇ、１２０μｍｏｌ、当量：１．１）、Ｎ−メチルモルホリン（１１０ｍｇ、１２０μｌ、１．０９ｍｍｏｌ、当量：１０）及びＥｔＯＡｃ（１０４ｍｇ、９７．２μｌ、３２６μｍｏｌ、当量：３）中Ｔ３Ｐ ５０％と室温で一晩撹拌した。飽和ＮａＨＣＯ_３溶液を添加し、酢酸エチルで３回抽出し、硫酸ナトリウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−５％メタノール）を使用したカラムクロマトグラフィーにより精製し、（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（４−（３−（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキサミド）−４−フルオロベンズアミド）フェニル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（７５ｍｇ、５１．５％）をオフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１３３７．６８ c) tert-butyl (2R, 5S) -4- [2- [3-[[4-[[3-[[(1S) -2-[(2S) -2-[[(2S) -2-] [Tert-Butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-1-carbonyl] amino] -4-fluoro-benzoyl] amino] phenyl] carbamoyl] -6 -[(4-Fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] Methyl] piperazin-1-carboxylate

tert-butyl ((S) -1-(((S) -2-((S) -1-((5-((4-aminophenyl) carbamoyl) -2-fluorophenyl) carbamoyl) isoindrin-2) -Il) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (78 mg, 109 μmol, equivalent: 1), 1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazine in ethyl acetate (1.5 ml)) -1-yl) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylic acid (76.5 mg, 120 μmol, equivalent: 1.1), N-methylmorpholin (110 mg, 120 μl, 1) Stirred overnight at room temperature with 50% T3P in .09 mmol, equivalent: 10) and EtOAc (104 mg, 97.2 μl, 326 μmol, equivalent: 3). Saturated NaHCO ₃ solution was added, extracted 3 times with ethyl acetate, dried over sodium sulfate, filtered and evaporated. The crude material was applied to silica gel and purified by column chromatography using ethyl acetate / methanol (0-5% methanol) as eluent and purified by (2R, 5S) -tert-butyl 4- (2- (3- (3- (3). (4- (3-((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-) Piran-4-yl) acetyl) isoindrin-1-carboxamide) -4-fluorobenzamide) phenyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl)- 2-Methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (75 mg, 51.5%) was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 1337.68

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（４−（３−（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキサミド）−４−フルオロベンズアミド）フェニル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（６５ｍｇ、４８．６μｍｏｌ、当量：１）を、酢酸エチル（１ｍｌ）中でジオキサン ４Ｍ （１ｍｌ、４ｍｍｏｌ、当量：８２．３）中ＨＣｌと室温で一晩撹拌した。沈殿した固形物を濾別し、ジエチルエーテルで洗浄した。固体を高減圧下で乾燥させ、Ｎ−（４−（４−フルオロ−３−（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキサミド）ベンズアミド）フェニル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド トリヒドロクロリド（５６ｍｇ、９２．４％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１１３７．５８ d) N- [4-[[4-fluoro-3-[[(1S) -2-[(2S) -2-[[(2S) -2- (methylamino) propanoyl] amino] -2-tetrahydro Piran-4-yl-acetyl] isoindolin-1-carbonyl] amino] benzoyl] amino] phenyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) ) -5-Methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide trihydrochloride

(2R, 5S) -tert-butyl 4-(2-(3-((4- (3-((S) -2-((S) -2-((S) -2-((tert-butoxy) Carbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-1-carboxamide) -4-fluorobenzamide) phenyl) carbamoyl) -6- (4-fluoro) Benzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (65 mg, 48.6 μmol, Equivalents: 1) were stirred overnight with HCl in dioxane 4M (1 ml, 4 mmol, equivalent: 82.3) in ethyl acetate (1 ml) at room temperature. The precipitated solid was filtered off and washed with diethyl ether. The solid was dried under high pressure and N- (4- (4-fluoro-3-((S) -2-((S) -2-((S) -2- (methylamino) propanoamide)-)- 2- (Tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-1-carboxamide) benzamide) phenyl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) )-5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride (56 mg, 92.4%) as a white solid Obtained. MS: m / z (M + H) ⁺ = 1137.58

ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（１００ｍｇ、１６２μｍｏｌ、当量：１、中間体２）を、ＥｔＯＡｃ ５０％（２５８ｍｇ、２４２μｌ、４０６μｍｏｌ、当量：２．５）中Ｔ３Ｐ及びピリジン（３００μｌ）の存在下、酢酸エチル（６００μｌ）中で３−（（（ベンジルオキシ）カルボニル）アミノ）プロパン酸（３６．３ｍｇ、１６２μｍｏｌ、当量：１）と室温で一晩撹拌した。飽和ＮａＨＣＯ３溶液を添加し、酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−５％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−（３−（（（ベンジルオキシ）カルボニル）アミノ）プロパンアミド）−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（１１８ｍｇ、８８．５％）を明黄色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝８２１．４ Example 17
a) (S) -N- (3-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) ) Propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-5-yl) amino) -3-oxopropyl) -6- (4-fluorobenzyl) -3-methyl-1 -(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride tert-butyl N -[(1S) -2-[[(1S) -2-[(1S) -5-[3- (benzylamino) propanoylamino] -1-[(2,6-difluorophenyl) carbamoyl] isoindrin -2-yl] -2-oxo-1-tetrahydropyran-4-yl-ethyl] amino] -1-methyl-2-oxo-ethyl] -N-methyl-carbamate

tert-butyl ((S) -1-(((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindrin-2-yl) -2-) Oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (100 mg, 162 μmol, equivalent: 1, intermediate 2), EtOAc 50 3-(((benzyloxy) carbonyl) amino) propanoic acid (36.3 mg) in ethyl acetate (600 μl) in the presence of T3P and pyridine (300 μl) in% (258 mg, 242 μl, 406 μmol, equivalent: 2.5). , 162 μmol, equivalent: 1) and stirred overnight at room temperature. Saturated NaHCO3 solution was added, extracted 3 times with ethyl acetate, dried over magnesium sulphate, filtered and evaporated. The crude substance was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-5% methanol) as an eluent, and tert-butyl ((S) -1-(((S) -2-)). ((S) -6-(3-(((benzyloxy) carbonyl) amino) propanamide) -1-((2,6-difluorophenyl) carbamoyl) isoindolin-2-yl) -2-oxo-1 -(Tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (118 mg, 88.5%) was obtained as a bright yellow solid. MS: m / z (M + H) ⁺ = 821.4

ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−１−（（２，６−ジフルオロフェニル）カルバモイル）−６−（３−（２−フェニルアセトアミド）プロパンアミド）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（１８０ｍｇ、２２４μｍｏｌ、当量：１）を、水素雰囲気下メタノール（８ｍｌ）中でパラジウム炭素１０％（２３．８ｍｇ、２２．４μｍｏｌ、当量：０．１）と室温で一晩撹拌した。触媒を濾別し、溶媒を真空中でエバポレートし、高減圧下で乾燥させて、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−（３−アミノプロパンアミド）−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（９３ｍｇ、６０．６％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６８７．３ b) tert-Butyl N-[(1S) -2-[[(1S) -2-[(1S) -6- (3-aminopropanoylamino) -1-[(2,6-difluorophenyl) carbamoyl) ] Isoindoline-2-yl] -2-oxo-1-tetrahydropyran-4-yl-ethyl] amino] -1-methyl-2-oxo-ethyl] -N-methyl-carbamate

tert-butyl ((S) -1-(((S) -2-((S) -1-((2,6-difluorophenyl) carbamoyl) -6- (3- (2-phenylacetamide) propanamide) ) Isoindoline-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (180 mg, 224 μmol, equivalent) 1) was stirred overnight at room temperature with 10% palladium carbon (23.8 mg, 22.4 μmol, equivalent: 0.1) in methanol (8 ml) under a hydrogen atmosphere. The catalyst is filtered off, the solvent is evaporated in vacuum and dried under high reduced pressure to allow tert-butyl ((S) -1-(((S) -2-((S) -6- (3-). Aminopropanamide) -1-((2,6-difluorophenyl) carbamoyl) isoindoline-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1- Oxopropan-2-yl) (methyl) carbamate (93 mg, 60.6%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 687.3

ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−（３−アミノプロパンアミド）−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（８７ｍｇ、１２７μｍｏｌ、当量：１）を、酢酸エチル（７００μｌ）及びピリジン（３５０μｌ）中で、（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（８０．９ｍｇ、１２７μｍｏｌ、当量：１）、ＥｔＯＡｃ ５０％（１０１ｍｇ、９４．３μｌ、３１７μｍｏｌ、当量：２．５）中Ｔ３Ｐと室温で一晩撹拌した。飽和ＮａＨＣＯ３溶液を添加し、酢酸エチルで３回抽出し、硫酸ナトリウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−５％メタノール）を使用したカラムクロマトグラフィーにより精製し、（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（３−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−３−オキソプロピル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（７０ｍｇ、４２．３％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１３０７．７ c) tert-butyl (2R, 5S) -4- [2-[(3S) -3-[[3-[[(3S) -2-[(2S) -2-[[(2S) -2-] [Tart-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino]- 3-oxo-propyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R)) -3-Methylmorpholin-4-yl] Methyl] piperazin-1-carboxylate

tert-butyl ((S) -1-(((S) -2-((S) -6- (3-aminopropanamide) -1-((2,6-difluorophenyl) carbamoyl) isoindrin-2) -Il) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (87 mg, 127 μmol, equivalent: 1), In ethyl acetate (700 μl) and pyridine (350 μl), (S) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R)-)- 3-Methylmorpholino) Methyl) Piperazin-1-yl) Acetyl) -6- (4-Fluorobenzyl) -3-Methylindrin-3-carboxylic Acid (80.9 mg, 127 μmol, Equivalent: 1), EtOAc 50% ( The mixture was stirred overnight at room temperature with T3P in 101 mg, 94.3 μl, 317 μmol, equivalent: 2.5). Saturated NaHCO3 solution was added, extracted 3 times with ethyl acetate, dried over sodium sulfate, filtered and evaporated. The crude material was applied to silica gel and purified by column chromatography using ethyl acetate / methanol (0-5% methanol) as eluent and purified by (2R, 5S) -tert-butyl 4-(2-((S)). -3-((3-(((S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-) 2H-pyran-4-yl) acetyl) -3-((2,6-difluorophenyl) carbamoyl) isoindolin-5-yl) amino) -3-oxopropyl) carbamoyl) -6- (4-fluorobenzyl) -3-Methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (70 mg, 42.3%) Obtained as a white solid. MS: m / z (M + H) ⁺ = 1307.7

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（３−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−３−オキソプロピル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（６４ｍｇ、４８．９μｍｏｌ、当量：１）を酢酸エチル（１ｍｌ）中でジオキサン ４Ｍ（１ｍｌ、４ｍｍｏｌ、当量：８１．７）中ＨＣｌと室温で一晩撹拌した。沈殿した固体を濾別し、ジエチルエーテルで洗浄し、高減圧下で乾燥させて、（Ｓ）−Ｎ−（３−（（（Ｓ）−３−（（２，６−ジフルオロフェニル）カルバモイル）−２−（（Ｓ）−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−５−イル）アミノ）−３−オキソプロピル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド トリヒドロクロリド（５５．５ｍｇ、４５．６μｍｏｌ、収率９３．２％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１１０７．５７ d) (3S) -N- [3-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methyl) Amino) propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] amino] -3-oxo-propyl] -6-[(4-fluorophenyl) methyl] -3-methyl -1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazine-1-yl] acetyl] indolin-3-carboxamide tri Hydrochloride

(2R, 5S) -tert-butyl 4-(2-((S) -3-((3-(((S) -2-((S) -2-((S) -2-((tert) -Butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) -3-((2,6-difluorophenyl) carbamoyl) isoindrin-5-yl) amino ) -3-oxopropyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) ) Methyl) piperazin-1-carboxylate (64 mg, 48.9 μmol, equivalent: 1) was stirred overnight in ethyl acetate (1 ml) with HCl in 4 M (1 ml, 4 mmol, equivalent: 81.7) of dioxane. .. The precipitated solid was filtered off, washed with diethyl ether and dried under high reduced pressure to (S) -N- (3-(((S) -3-((2,6-difluorophenyl) carbamoyl)). -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) amino)- 3-oxopropyl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl)) Piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride (55.5 mg, 45.6 μmol, yield 93.2%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1107.57

ベンジル ４−アミノブタノエート（２８．３ｍｇ、１４６μｍｏｌ、当量：１．１）を、酢酸エチル（０．８５ｍｌ）中（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（８５ｍｇ、１３３μｍｏｌ、当量：１）の明黄色の懸濁液に添加した。Ｎ−メチルモルホリン（１３５ｍｇ、１４６μｌ、１．３３ｍｍｏｌ、当量：１０）及びＥｔＯＡｃ（２５４ｍｇ、２３８μｌ、３９９μｍｏｌ、当量：３）中Ｔ３Ｐ ５０％を添加し、反応物を室温で一晩撹拌した。反応物を酢酸エチルに注ぎ、水で抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗残留物をシリカゲルに適用し、溶出剤として酢酸エチル／ヘプタン（８０−１００％酢酸エチル）を使用したフラッシュクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｓ）−３−［（４−ベンジルオキシ−４−オキソ−ブチル）カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（６７ｍｇ、６１．９％）を無色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝８１４．４６ Example 18
a) (S) -N- (4-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) ) Propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-5-yl) amino) -4-oxobutyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride tert-butyl (2R) , 5S) -4- [2-[(3S) -3-[(4-benzyloxy-4-oxo-butyl) carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indrin- 1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate

Benzyl 4-aminobutanoate (28.3 mg, 146 μmol, equivalent: 1.1) in ethyl acetate (0.85 ml) (S) -1-(2-((2S, 5R) -4- (tert) -Butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxylic acid It was added to a light yellow suspension of acid (85 mg, 133 μmol, equivalent: 1). 50% T3P in N-methylmorpholine (135 mg, 146 μl, 1.33 mmol, equivalent: 10) and EtOAc (254 mg, 238 μl, 399 μmol, equivalent: 3) was added and the reaction was stirred at room temperature overnight. The reaction was poured into ethyl acetate and extracted with water. The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude residue was applied to silica gel and purified by flash chromatography using ethyl acetate / heptane (80-100% ethyl acetate) as eluent and tert-butyl (2R, 5S) -4- [2-[( 3S) -3-[(4-benzyloxy-4-oxo-butyl) carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-Methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (67 mg, 61.9%) was obtained as a colorless oil. MS: m / z (M + H) ⁺ = 814.46

Ｐｄ−Ｃ（８．５ｍｇ、７．９９μｍｏｌ、当量：０．１）を、メタノール（０．５ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（４−（ベンジルオキシ）−４−オキソブチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（６５ｍｇ、７９．９μｍｏｌ、当量：１）の透明な溶液に添加し、反応物を水素雰囲気下室温で６時間撹拌した。触媒を濾別し、濾液をエバポレートして、４−［［（３Ｓ）−１−［２−［（２Ｓ，５Ｒ）−４−ｔｅｒｔ−ブトキシカルボニル−５−メチル−２−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−イル］アセチル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−３−カルボニル］アミノ］ブタン酸（５８ｍｇ、１００％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝７２４．４ b) 4-[[(3S) -1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R) -3-methylmorpholine-4-yl]] Methyl] piperazine-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carbonyl] amino] butanoic acid

Pd-C (8.5 mg, 7.9 μmol, equivalent: 0.1) in methanol (0.5 ml) (2R, 5S) -tert-butyl 4-(2-((S) -3-(((S) -3-((). 4- (Benzyloxy) -4-oxobutyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R)-)- It was added to a clear solution of 3-methylmorpholino) methyl) piperazin-1-carboxylate (65 mg, 79.9 μmol, equivalent: 1) and the reaction was stirred under a hydrogen atmosphere at room temperature for 6 hours. The catalyst was filtered off and the filtrate was evaporated to 4-[[(3S) -1- [2-[(2S, 5R) -4-tert-butoxycarbonyl-5-methyl-2-[[(3R)). -3-Methylmorpholin-4-yl] methyl] piperazine-1-yl] acetyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-3-carbonyl] amino] butanoic acid (58 mg, 100%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 724.4

ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（４６．８ｍｇ、７６μｍｏｌ、当量：１；中間体２）、ピリジン（３００μｌ）及びＥｔＯＡｃ（１４５ｍｇ、１３６μｌ、２２８μｍｏｌ、当量：３）中Ｔ３Ｐ ５０％を、酢酸エチル（０．６ｍｌ）中４−（（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）ブタン酸（５５ｍｇ、７６μｍｏｌ、当量：１）の白色の懸濁液に添加し、明黄色の溶液を室温で一晩撹拌した。反応物を数滴のａｑ． ＨＣｌ １Ｎでクエンチし、酢酸エチル及び水で抽出した。有機層を、硫酸ナトリウムで乾燥させ、濾過し、真空中でエバポレートした。粗残留物をシリカゲルに適用し、溶出剤としてジクロロメタン／メタノール（０−２０％メタノール）を使用したフラッシュクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｓ）−３−［［４−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］アミノ］−４−オキソ−ブチル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（１３ｍｇ、１２．９％）を白色の非晶質として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１３２１．７ c) tert-Butyl (2R, 5S) -4- [2-[(3S) -3-[[4-[[(3S) -2-[(2S) -2-[[(2S) -2-] [Tert-Butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino]- 4-Oxo-butyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R)) -3-Methylmorpholin-4-yl] Methyl] piperazin-1-carboxylate

tert-butyl ((S) -1-(((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindrin-2-yl) -2-) Oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (46.8 mg, 76 μmol, equivalent: 1; intermediate 2), pyridine 50% T3P in (300 μl) and EtOAc (145 mg, 136 μl, 228 μmol, equivalent: 3), 4-((S) -1- (2-((2S, 5R) -4) in ethyl acetate (0.6 ml)). -(Tert-Butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6- (4-fluorobenzyl) -3-methylindrin- 3-Carboxamide) Butanoic acid (55 mg, 76 μmol, equivalent: 1) was added to a white suspension and the bright yellow solution was stirred at room temperature overnight. Add a few drops of the reaction to aq. It was quenched with HCl 1N and extracted with ethyl acetate and water. The organic layer was dried over sodium sulphate, filtered and evaporated in vacuo. The crude residue was applied to silica gel and purified by flash chromatography using dichloromethane / methanol (0-20% methanol) as eluent and tert-butyl (2R, 5S) -4- [2-[(3S)). -3-[[4-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran- 4-Il-acetyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino] -4-oxo-butyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-Methyl-Indoline-1-yl] -2-oxo-ethyl] -2-Methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (13 mg) , 12.9%) was obtained as white amorphous. MS: m / z (M + H) ⁺ = 1321.7

ジオキサン（２０８μｌ、８３２μｍｏｌ、当量：１００）中ＨＣｌ ４Ｍを、酢酸エチル（０．２ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（４−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−４−オキソブチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（１１ｍｇ、８．３２μｍｏｌ、当量：１）の明黄色の溶液に添加し、反応物を室温で２時間撹拌した。ジエチルエーテルを添加し、白色の沈殿物を濾過し、ジエチルエーテルで洗浄し、乾燥させて、（Ｓ）−Ｎ−（４−（（（Ｓ）−３−（（２，６−ジフルオロフェニル）カルバモイル）−２−（（Ｓ）−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−５−イル）アミノ）−４−オキソブチル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド トリヒドロクロリド（９．４ｍｇ、９１．８％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１１２１．６ d) (3S) -N- [4-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methyl) Amino) propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] amino] -4-oxo-butyl] -6-[(4-fluorophenyl) methyl] -3-methyl -1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide tri Hydrochloride

HCl 4M in dioxane (208 μl, 832 μmol, equivalent: 100) in ethyl acetate (0.2 ml) (2R, 5S) -tert-butyl 4-(2-((S) -3-((4-(((). (S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl ) -3-((2,6-difluorophenyl) carbamoyl) isoindrin-5-yl) amino) -4-oxobutyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) Add to a bright yellow solution of -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (11 mg, 8.32 μmol, equivalent: 1). , The reaction was stirred at room temperature for 2 hours. Diethyl ether is added, the white precipitate is filtered, washed with diethyl ether, dried and (S) -N- (4-(((S) -3-((2,6-difluorophenyl))). Carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) amino ) -4-oxobutyl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) ) Piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride (9.4 mg, 91.8%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1121.6

２−（（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（５０ｍｇ、７１．９μｍｏｌ、当量：１）を、酢酸エチル（５００μｌ）及びピリジン（２５０μｌ）中で、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−５−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（４４．２ｍｇ、７１．９μｍｏｌ、当量：１；中間体４）及びＥｔＯＡｃ ５０％（１３７ｍｇ、１２８μｌ、２１６μｍｏｌ、当量：３）中Ｔ３Ｐと室温で一晩撹拌した。飽和ＮａＨＣＯ３溶液を添加し、酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−１０％メタノール）を使用したカラムクロマトグラフィーにより精製した。分離された生成物をさらなる精製のために分取ＨＰＬＣに供し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｓ）−３−［［２−［［（１Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−２−テトラヒドロピラン−４−イル−アセチル］−１−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］アミノ］−２−オキソ−エチル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（７ｍｇ、７．５％）を無色の非晶質として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２９３．８ Example 19
a) (S) -N- (2-(((S) -1-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) ) Propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride tert-butyl (2R) , 5S) -4- [2-[(3S) -3-[[2-[[(1S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) ) Amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -1-[(2,6-difluorophenyl) carbamoyl] isoindolin-5-yl] amino] -2-oxo-ethyl] carbamoyl ] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4 -Il] Methyl] Piperazin-1-carboxylate

2-((S) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxamide) Acetic acid (50 mg, 71.9 μmol, equivalent: 1) in ethyl acetate (500 μl) and pyridine (250 μl). , Tert-Butyl ((S) -1-(((S) -2-((S) -5-amino-1-((2,6-difluorophenyl) carbamoyl) isoindrin-2-yl) -2) -Oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (44.2 mg, 71.9 μmol, equivalent: 1; intermediate 4 ) And EtOAc in 50% (137 mg, 128 μl, 216 μmol, equivalent: 3) with T3P and stirred overnight at room temperature. Saturated NaHCO3 solution was added, extracted 3 times with ethyl acetate, dried over magnesium sulphate, filtered and evaporated. The crude material was applied to silica gel and purified by column chromatography using ethyl acetate / methanol (0-10% methanol) as the eluent. The separated products were subjected to preparative HPLC for further purification and tert-butyl (2R, 5S) -4- [2-[(3S) -3-[[2-[[(1S) -2-]. [(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] -1-[(2,6-] Difluorophenyl) carbamoyl] isoindrin-5-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indrin-1-yl] -2-oxo -Ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (7 mg, 7.5%) was obtained as a colorless amorphous substance. .. MS: m / z (M + H) ⁺ = 1293.8

ジオキサン（１３５μｌ、５４１μｍｏｌ、当量：１００）中ＨＣｌ ４Ｍを、酢酸エチル（２００μｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（７ｍｇ、５．４１μｍｏｌ、当量：１）の無色の溶液に添加し、反応物を室温で一晩撹拌した。沈殿物を濾別し、ジエチルエーテルで洗浄し、真空中で乾燥させて、（Ｓ）−Ｎ−（２−（（（Ｓ）−１−（（２，６−ジフルオロフェニル）カルバモイル）−２−（（Ｓ）−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−５−イル）アミノ）−２−オキソエチル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド トリヒドロクロリド（３．７ｍｇ、５６．９％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０９３．５４ b) (3S) -N- [2-[[(1S) -1-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methyl) Amino) propanoyl] amino] -2-tetrahydropyran-4-yl-acetyl] isoindolin-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl -1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide tri Hydrochloride

HCl 4M in dioxane (135 μl, 541 μmol, equivalent: 100) and (2R, 5S) -tert-butyl 4-(2-((S) -3-((2-(((S))) in ethyl acetate (200 μl). ) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl)- 1-((2,6-difluorophenyl) carbamoyl) isoindrin-5-yl) amino) -2-oxoethyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2 -Oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (7 mg, 5.41 μmol, equivalent: 1) was added to a colorless solution and the reaction product was added. Was stirred overnight at room temperature. The precipitate was filtered off, washed with diethyl ether, dried in vacuo and (S) -N- (2-(((S) -1-((2,6-difluorophenyl) carbamoyl) -2). -((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-5-yl) amino) -2- Oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) Acetyl) Indoline-3-Carboxamide Trihydrochloride (3.7 mg, 56.9%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1093.54

２−（（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（７０ｍｇ、１０１μｍｏｌ、当量：１）を、酢酸エチル（７００μｌ）及びピリジン（３５０μｌ）中で、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（２Ｓ，３Ｒ）−１−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−３−メトキシ−１−オキソブタン−２−イル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（５９．３ｍｇ、１０１μｍｏｌ、当量：１；中間体５）及びＥｔＯＡｃ ５０％（１９２ｍｇ、１８０μｌ、３０２μｍｏｌ、当量：３）中Ｔ３Ｐと室温で一晩撹拌した。反応混合物をＨＣｌ ａｑ． １Ｎ、飽和ＮａＨＣＯ_３及びブラインで抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−２０％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｓ）−３−［［２−［［（３Ｓ）−２−［（２Ｓ，３Ｒ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−３−メトキシ−ブタノイル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］アミノ］−２−オキソ−エチル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（１００ｍｇ、７８．４％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２６７．６ Example 20
(S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((2S, 3R) -3-methoxy-2-((S) -2) -(Methylamino) propanamide) butanoyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)-)- 5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride a) tert-butyl (2R, 5S) -4- [2 -[(3S) -3-[[2-[[(3S) -2-[(2S, 3R) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] ] -3-Methyl-butanoyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) Methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate

2-((S) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxamide) tert Acetic acid (70 mg, 101 μmol, equivalent: 1) in ethyl acetate (700 μl) and pyridine (350 μl). -Butyl ((S) -1-(((2S, 3R) -1-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindrin-2-yl) -3 −methoxy-1-oxobutan-2-yl) amino) -1-oxopropan-2-yl) (methyl) carbamate (59.3 mg, 101 μmol, equivalent: 1; intermediate 5) and EtOAc 50% (192 mg, 180 μl) , 302 μmol, equivalent: 3) T3P in medium and stirred overnight at room temperature. Hydrochloric acid aq. Extracted with 1N, saturated NaHCO ₃ and brine. The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude material was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-20% methanol) as eluent, and tert-butyl (2R, 5S) -4- [2-[(3S)). -3-[[2-[[(3S) -2-[(2S, 3R) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -3-methoxy] -Butanoyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3- Methyl-indrin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (100 mg, 78. 4%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1267.6

ジオキサン（１．８７ｍｌ、７．５ｍｍｏｌ、当量：１００）中ＨＣｌ ４Ｍを、酢酸エチル（１ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（２−（（（Ｓ）−２−（（２Ｓ，３Ｒ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−３−メトキシブタノイル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（９５ｍｇ、７５μｍｏｌ、当量：１）の無色の溶液に添加した。反応物を室温で４５分間撹拌した。白色の沈殿物を濾別し、ジエチルエーテルで洗浄し、真空中で乾燥させて、（Ｓ）−Ｎ−（２−（（（Ｓ）−３−（（２，６−ジフルオロフェニル）カルバモイル）−２−（（２Ｓ，３Ｒ）−３−メトキシ−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）ブタノイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド トリヒドロクロリド （７６ｍｇ、８６．２％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０６７．５ b) (3S) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S, 3R) -3-methoxy-2-[[(2S) ) -2- (Methylamino) propanoyl] amino] butanoyl] isoindrin-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [ 2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazine-1-yl] acetyl] indolin-3-carboxamide trihydrochloride

HCl 4M in dioxane (1.87 ml, 7.5 mmol, equivalent: 100) and (2R, 5S) -tert-butyl 4-(2-((S) -3-((2-)) in ethyl acetate (1 ml)). (((S) -2-((2S, 3R) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -3-methoxybutanoyl) -3-( (2,6-difluorophenyl) carbamoyl) isoindrin-5-yl) amino) -2-oxoethyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) It was added to a colorless solution of -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (95 mg, 75 μmol, equivalent: 1). The reaction was stirred at room temperature for 45 minutes. The white precipitate was filtered off, washed with diethyl ether, dried in vacuo and (S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl)). -2-((2S, 3R) -3-methoxy-2-((S) -2- (methylamino) propanamide) butanoyl) isoindolin-5-yl) amino) -2-oxoethyl) -6-( 4-Fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin -3-Carboxamide trihydrochloride (76 mg, 86.2%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1067.5

２−（（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（４０ｍｇ、５７．５μｍｏｌ、当量：１）を、酢酸エチル（０．５ｍｌ）及びピリジン（２５０μｌ）中で、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（２Ｓ，３Ｓ）−１−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−３−メチル−１−オキソペンタン−２−イル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（３３．８ｍｇ、５７．５μｍｏｌ、当量：１；中間体６）及びＥｔＯＡｃ ５０％（１１０ｍｇ、１０３μｌ、１７２μｍｏｌ、当量：３）中Ｔ３Ｐと室温で一晩撹拌した。反応混合物を飽和水性ＮａＨＣＯ_３及びブラインで抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−２０％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｓ）−３−［［２−［［（３Ｓ）−２−［（２Ｓ，３Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−３−メチル−ペンタノイル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］アミノ］−２−オキソ−エチル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（４１ｍｇ、５６．４％）を黄色の非晶質として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２６５．７ Example 21
a) (S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((2S, 3S) -3-methyl-2-((S)) -2- (Methylamino) propanamide) pentanoyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) ) -5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride tert-butyl (2R, 5S) -4- [2 -[(3S) -3-[[2-[[(3S) -2-[(2S, 3S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] ] -3-Methyl-pentanoyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) Methyl] -3-methyl-indrin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate

2-((S) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxamide) Acetic acid (40 mg, 57.5 μmol, equivalent: 1), ethyl acetate (0.5 ml) and pyridine (250 μl) Among them, tert-butyl ((S) -1-(((2S, 3S) -1-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindrin-2- Ill) -3-methyl-1-oxopentan-2-yl) amino) -1-oxopropan-2-yl) (methyl) carbamate (33.8 mg, 57.5 μmol, equivalent: 1; intermediate 6) and Stir overnight with T3P in 50% EtOAc (110 mg, 103 μl, 172 μmol, equivalent: 3) at room temperature. The reaction mixture was extracted with _{saturated aqueous NaHCO 3 and brine.} The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude material was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-20% methanol) as eluent, and tert-butyl (2R, 5S) -4- [2-[(3S)). -3-[[2-[[(3S) -2-[(2S, 3S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -3-methyl -Pentanoyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3- Methyl-indrin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (41 mg, 56. 4%) was obtained as yellow amorphous. MS: m / z (M + H) ⁺ = 1265.7

ジオキサン（６９１μｌ、２．７７ｍｍｏｌ、当量：１００）中ＨＣｌ ４Ｍを、酢酸エチル（０．５ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（２−（（（Ｓ）−２−（（２Ｓ，３Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−３−メチルペンタノイル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（３５ｍｇ、２７．７μｍｏｌ、当量：１）の明黄色の溶液に添加し、反応物を室温で２時間撹拌した。オフホワイトの沈殿物を濾別し、ジエチルエーテルで洗浄し、真空中で乾燥させて、（Ｓ）−Ｎ−（２−（（（Ｓ）−３−（（２，６−ジフルオロフェニル）カルバモイル）−２−（（２Ｓ，３Ｓ）−３−メチル−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）ペンタノイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド トリヒドロクロリド（２５ｍｇ、７７％）をオフホワイトの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０６５．５５ b) (3S) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S, 3S) -3-methyl-2-[[(2S) ) -2- (Methylamino) propanoyl] amino] pentanoyl] isoindrin-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [ 2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indolin-3-carboxamide trihydrochloride

HCl 4M in dioxane (691 μl, 2.77 mmol, equivalent: 100) and (2R, 5S) -tert-butyl 4-(2-((S) -3-((2-)) in ethyl acetate (0.5 ml)). (((S) -2-((2S, 3S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -3-methylpentanoyl) -3-( (2,6-difluorophenyl) carbamoyl) isoindrin-5-yl) amino) -2-oxoethyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) Add -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (35 mg, 27.7 μmol, equivalent: 1) to a bright yellow solution and bring the reaction to room temperature. Was stirred for 2 hours. The off-white precipitate was filtered off, washed with diethyl ether, dried in vacuo and (S) -N-(2-(((S) -3-((2,6-difluorophenyl) carbamoyl)). ) -2-((2S, 3S) -3-methyl-2-((S) -2- (methylamino) propanamide) pentanoyl) isoindoline-5-yl) amino) -2-oxoethyl) -6- (4-Fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) Indoline-3-carboxamide trihydrochloride (25 mg, 77%) was obtained as an off-white solid. MS: m / z (M + H) ⁺ = 1065.55

２−（（Ｓ）−１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキサミド）酢酸（７１ｍｇ、１０２μｍｏｌ、当量：１）を、酢酸エチル（８００μｌ）及びピリジン（４００μｌ）中で、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−１−（（Ｓ）−６−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−３，３−ジメチル−１−オキソｂｕｔａｎ−２−イル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（６０ｍｇ、１０２μｍｏｌ、当量：１；中間体７）及びＥｔＯＡｃ ５０％（１９５ｍｇ、１８２μｌ、３０６μｍｏｌ、当量：３）中Ｔ３Ｐと室温で一晩撹拌した。反応混合物をＨＣｌ ａｑ． １Ｎ、飽和ＮａＨＣＯ_３及びブラインで抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−５％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［（３Ｓ）−３−［［２−［［（３Ｓ）−２−［（２Ｓ）−２−［［（２Ｓ）−２−［ｔｅｒｔ−ブトキシカルボニル（メチル）アミノ］プロパノイル］アミノ］−３，３−ジメチル−ブタノイル］−３−［（２，６−ジフルオロフェニル）カルバモイル］イソインドリン−５−イル］アミノ］−２−オキソ−エチル］カルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（８６ｍｇ、６６．６％）を得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１２６５．７ Example 22
a) (S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -3,3-dimethyl-2-((S)) -2- (Methylamino) Propanamide) Butanoyl) Isoindrin-5-yl) Amino) -2-oxoethyl) -6- (4-Fluorobenzyl) -3-Methyl-1- (2-((2R, 5R) ) -5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride tert-butyl (2R, 5S) -4- [2 -[(3S) -3-[[2-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino]- 3,3-Dimethyl-butanoyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) Methyl] -3-methyl-indrin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate

2-((S) -1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxamide) tert Acetic acid (71 mg, 102 μmol, equivalent: 1) in ethyl acetate (800 μl) and pyridine (400 μl). -Butyl ((S) -1-(((S) -1-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindrin-2-yl) -3,3 -Dimethyl-1-oxobutan-2-yl) amino) -1-oxopropan-2-yl) (methyl) carbamate (60 mg, 102 μmol, equivalent: 1; intermediate 7) and EtOAc 50% (195 mg, 182 μl, 306 μmol, equivalent: 3) T3P in medium and stirred overnight at room temperature. Hydrochloric acid aq. Extracted with 1N, saturated NaHCO ₃ and brine. The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude substance was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-5% methanol) as eluent, and tert-butyl (2R, 5S) -4- [2-[(3S)). -3-[[2-[[(3S) -2-[(2S) -2-[[(2S) -2- [tert-butoxycarbonyl (methyl) amino] propanoyl] amino] -3,3-dimethyl -Butanoyl] -3-[(2,6-difluorophenyl) carbamoyl] isoindrin-5-yl] amino] -2-oxo-ethyl] carbamoyl] -6-[(4-fluorophenyl) methyl] -3- Methyl-indoline-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (86 mg, 66. 6%) was obtained. MS: m / z (M + H) ⁺ = 1265.7

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（（Ｓ）−３−（（２−（（（Ｓ）−２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−３，３−ジメチルブタノイル）−３−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（８０ｍｇ、６３．２μｍｏｌ、当量：１）を、酢酸エチル（１ｍｌ）中でジオキサン ４Ｍ（１ｍｌ、４ｍｍｏｌ、当量：６３．３）中ＨＣｌと室温で２時間撹拌した。沈殿した固体を濾別し、ジエチルエーテルで洗浄し、高減圧下で乾燥させて、（Ｓ）−Ｎ−（２−（（（Ｓ）−３−（（２，６−ジフルオロフェニル）カルバモイル）−２−（（Ｓ）−３，３−ジメチル−２−（（Ｓ）−２−（メチルアミノ）プロパンアミド）ブタノイル）イソインドリン−５−イル）アミノ）−２−オキソエチル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド トリヒドロクロリド （４５ｍｇ、６０．６％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝１０６５．５５ b) (3S) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -3,3-dimethyl-2-[[(2S) ) -2- (Methylamino) propanoyl] amino] butanoyl] isoindoline-5-yl] amino] -2-oxo-ethyl] -6-[(4-fluorophenyl) methyl] -3-methyl-1- [ 2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-yl] acetyl] indoline-3-carboxamide trihydrochloride

(2R, 5S) -tert-butyl 4-(2-((S) -3-((2-(((S) -2-((S) -2-((S) -2-((tert) -Butoxycarbonyl) (methyl) amino) propanamide) -3,3-dimethylbutanoyl) -3-((2,6-difluorophenyl) carbamoyl) isoindolin-5-yl) amino) -2-oxoethyl) carbamoyl ) -6- (4-Fluorobenzyl) -3-methylindrin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxy The rate (80 mg, 63.2 μmol, equivalent: 1) was stirred in ethyl acetate (1 ml) with HCl in 4 M (1 ml, 4 mmol, equivalent: 63.3) of dioxane at room temperature for 2 hours. The precipitated solid was filtered off, washed with diethyl ether and dried under high reduced pressure to (S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl)). -2-((S) -3,3-dimethyl-2-((S) -2- (methylamino) propanamide) butanoyl) isoindoline-5-yl) amino) -2-oxoethyl) -6-( 4-Fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indoline -3-Carboxamide trihydrochloride (45 mg, 60.6%) was obtained as a white solid. MS: m / z (M + H) ⁺ = 1065.55

１−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボン酸（３００ｍｇ、４７０μｍｏｌ、当量：１）を、ＤＩＰＥＡ（２４３ｍｇ、３２８μｌ、１．８８ｍｍｏｌ、当量：４）及びＨＡＴＵ（２３２ｍｇ、６１１μｍｏｌ、当量：１．３）と室温で一晩撹拌した。反応混合物を０．５Ｍ ＨＣｌ ａｑ．に注ぎ、酢酸エチルで３回抽出し、ブラインで洗浄し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗物質をシリカゲルに適用し、溶出剤として酢酸エチル／メタノール（０−５％メタノール）を使用したカラムクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［３−［２−（ベンジルオキシカルボニルアミノ）エチルカルバモイル］−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（４８２ｍｇ、１００％）を明黄色の油として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝８１５．４５ Monomer A
N- (2-aminoethyl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) ) Methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide dihydrochloride a) tert-butyl (2R, 5S) -4- [2- [3- [2- (benzyloxycarbonylamino) ethylcarbamoyl]- 6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholin-4-yl] ] Methyl] piperazin-1-carboxylate

1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6 -(4-Fluorobenzyl) -3-methylindrin-3-carboxylic acid (300 mg, 470 μmol, equivalent: 1), DIPEA (243 mg, 328 μl, 1.88 mmol, equivalent: 4) and HATU (232 mg, 611 μmol, equivalent). : 1.3) and stirred overnight at room temperature. The reaction mixture was added to 0.5 M HCl aq. Pour into, extracted 3 times with ethyl acetate, washed with brine, dried over magnesium sulphate, filtered and evaporated. The crude material was applied to silica gel, purified by column chromatography using ethyl acetate / methanol (0-5% methanol) as eluent, and tert-butyl (2R, 5S) -4- [2- [3- [ 2- (benzyloxycarbonylamino) ethyl carbamoyl] -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[[ (3R) -3-Methylmorpholin-4-yl] methyl] piperazine-1-carboxylate (482 mg, 100%) was obtained as a bright yellow oil. MS: m / z (M + H) ⁺ = 815.45

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（２−（（（ベンジルオキシ）カルボニル）アミノ）エチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（３７５ｍｇ、４６０μｍｏｌ、当量：１）を、水素雰囲気下メタノール（１５ｍｌ）中でパラジウム炭素１０％（４９ｍｇ、４６μｍｏｌ、当量：０．１）と室温で一晩撹拌した。５滴の酢酸を添加し、水素雰囲気下室温で撹拌を３時間続けた。触媒を濾別し、溶媒をエバポレートした。重炭酸水素ナトリウム溶液を添加し、混合物を酢酸エチルで３回抽出し、硫酸マグネシウムで乾燥させ、濾過し、エバポレートして、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［３−（２−アミノエチルカルバモイル）−６−［（４−フルオロフェニル）メチル］−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（２８１ｍｇ、８９．７％）を明黄色の泡として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６８１．４２ b) tert-Butyl (2R, 5S) -4- [2- [3- (2-aminoethylcarbamoyl) -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl]- 2-Oxo-ethyl] -2-methyl-5-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazine-1-carboxylate

(2R, 5S) -tert-butyl 4-(2-(3-((2-(((benzyloxy) carbonyl) amino) ethyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindrin- 1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazin-1-carboxylate (375 mg, 460 μmol, equivalent: 1) in methanol under a hydrogen atmosphere Stirred overnight in (15 ml) with 10% palladium carbon (49 mg, 46 μmol, equivalent: 0.1) at room temperature. Five drops of acetic acid were added and stirring was continued for 3 hours at room temperature under a hydrogen atmosphere. The catalyst was filtered off and the solvent was evaporated. Sodium bicarbonate solution is added, the mixture is extracted 3 times with ethyl acetate, dried over magnesium sulfate, filtered, evaporated and tert-butyl (2R, 5S) -4- [2- [3-( 2-Aminoethyl carbamoyl) -6-[(4-fluorophenyl) methyl] -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3 -Methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (281 mg, 89.7%) was obtained as a bright yellow foam. MS: m / z (M + H) ⁺ = 681.42

（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（３−（（２−アミノエチル）カルバモイル）−６−（４−フルオロベンジル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（５０ｍｇ、７３．４μｍｏｌ、当量：１）を、酢酸エチル（１ｍｌ）中でジオキサン４Ｍ（１ｍｌ、４ｍｍｏｌ、当量：５４．５）中ＨＣｌと室温で一晩撹拌した。沈殿した固体を濾別し、ジエチルエーテルで洗浄し、高減圧下で乾燥させて、Ｎ−（２−アミノエチル）−６−（４−フルオロベンジル）−３−メチル−１−（２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）インドリン−３−カルボキサミド ジヒドロクロリド（３５．５ｍｇ、７４％）を白色の固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝５８１．３６ c) N- (2-aminoethyl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-3) Methylmorpholino) methyl) piperazine-1-yl) acetyl) indolin-3-carboxamide dihydrochloride

(2R, 5S) -tert-butyl 4-(2-(3-((2-aminoethyl) carbamoyl) -6- (4-fluorobenzyl) -3-methylindolin-1-yl) -2-oxoethyl) -2-Methyl-5-(((R) -3-methylmorpholino) methyl) piperazine-1-carboxylate (50 mg, 73.4 μmol, equivalent: 1) in ethyl acetate (1 ml) with dioxane 4M (1 ml) Stirred overnight at room temperature with HCl in 4, 4 mmol, equivalent: 54.5). The precipitated solid is filtered off, washed with diethyl ether and dried under high vacuum to allow N- (2-aminoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-( White (2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide dihydrochloride (35.5 mg, 74%) Obtained as a solid of. MS: m / z (M + H) ⁺ = 581.36

Monomer B
(S) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-) 1-yl) Acetyl) Indoline-3-carboxylic acid dihydrochloride dioxane (1.96 ml, 7.83 mmol, equivalent: 100) HCl 4M in ethyl acetate (0.5 ml) (S) -1- (2-) ((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) -6- (4-fluoro) It was added to a yellow suspension of benzyl) -3-methylindrin-3-carboxylic acid (50 mg, 78.3 μmol, equivalent: 1) and the reaction was stirred at room temperature for 3 hours. The white precipitate was filtered off, washed with diethyl ether, dried in vacuo and (S) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)). Obtained -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxylic acid dihydrochloride (30 mg, 62.7%) as an orange solid. rice field. MS: m / z (M + H) ⁺ = 539.3

水素化ホウ素ナトリウム（８５．８ｍｇ、２．２７ｍｍｏｌ、当量：２）を、ＴＨＦ（５．８６ｍｌ）及びメタノール（５．８６ｍｌ）中メチル １−（２−（（２Ｓ，５Ｒ）−４−（ｔｅｒｔ−ブトキシカルボニル）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）アセチル）−６−（４−フルオロベンジル）−３−メチルインドリン−３−カルボキシレート（７４０ｍｇ、１．１３ｍｍｏｌ、当量：１）の透明な溶液に０℃で添加した。反応物を０℃で５時間撹拌し、その後、室温まで温め、一晩撹拌した。Ａｑ． ｓａｔ． 炭酸ナトリウムを添加し、混合物を酢酸エチルで抽出した。有機層を、硫酸マグネシウムで乾燥させ、濾過し、エバポレートした。粗残留物をシリカゲルに適用し、溶出剤として酢酸エチル／ヘプタン（５０−１００％酢酸エチル）を使用したフラッシュクロマトグラフィーにより精製し、ｔｅｒｔ−ブチル（２Ｒ，５Ｓ）−４−［２−［６−［（４−フルオロフェニル）メチル］−３−（ヒドロキシメチル）−３−メチル−インドリン−１−イル］−２−オキソ−エチル］−２−メチル−５−［［（３Ｒ）−３−メチルモルホリン−４−イル］メチル］ピペラジン−１−カルボキシレート（６１６ｍｇ、８７％）を白色の泡として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６２５．４ Monomer C
1-(6- (4-Fluorobenzyl) -3- (hydroxymethyl) -3-methylindrin-1-yl) -2-((2R, 5R) -5-methyl-2-(((R)-)- 3-Methylmorpholino) Methyl) Piperazin-1-yl) Etanone dihydrochloride a) tert-butyl (2R, 5S) -4- [2- [6-[(4-fluorophenyl) methyl] -3- (hydroxymethyl) ) -3-Methyl-Indoline-1-yl] -2-oxo-ethyl] -2-Methyl-5-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazin-1-carboxylate

Sodium borohydride (85.8 mg, 2.27 mmol, equivalent: 2) in methyl 1- (2-((2S, 5R) -4- (tert)) in THF (5.86 ml) and methanol (5.86 ml). -Butoxycarbonyl) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazine-1-yl) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxy It was added to a clear solution of rate (740 mg, 1.13 mmol, equivalent: 1) at 0 ° C. The reaction was stirred at 0 ° C. for 5 hours, then warmed to room temperature and stirred overnight. Aq. sat. Sodium carbonate was added and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulphate, filtered and evaporated. The crude residue was applied to silica gel and purified by flash chromatography using ethyl acetate / heptane (50-100% ethyl acetate) as eluent and tert-butyl (2R, 5S) -4- [2- [6]. -[(4-Fluorophenyl) methyl] -3- (hydroxymethyl) -3-methyl-indolin-1-yl] -2-oxo-ethyl] -2-methyl-5-[[(3R) -3-] Methylmorpholin-4-yl] methyl] piperazin-1-carboxylate (616 mg, 87%) was obtained as white foam. MS: m / z (M + H) ⁺ = 625.4

ジオキサン（２ｍｌ、８ｍｍｏｌ、当量：１００）中ＨＣｌ ４Ｍを、酢酸エチル（０．５ｍｌ）中（２Ｒ，５Ｓ）−ｔｅｒｔ−ブチル ４−（２−（６−（４−フルオロベンジル）−３−（ヒドロキシメチル）−３−メチルインドリン−１−イル）−２−オキソエチル）−２−メチル−５−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−カルボキシレート（５０ｍｇ、８０μｍｏｌ、当量：１）の透明な溶液に添加し、反応混合物を室温で３時間撹拌した。沈殿物を濾別し、ジエチルエーテルで完全に洗浄し（非常に吸湿性がある）、真空中で乾燥させて、１−（６−（４−フルオロベンジル）−３−（ヒドロキシメチル）−３−メチルインドリン−１−イル）−２−（（２Ｒ，５Ｒ）−５−メチル−２−（（（Ｒ）−３−メチルモルホリノ）メチル）ピペラジン−１−イル）エタノンジヒドロクロリド （４３ｍｇ、８９．９ ％）をオレンジの固体として得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝５２５．３ b) 1- [6-[(4-fluorophenyl) methyl] -3- (hydroxymethyl) -3-methyl-indolin-1-yl] -2-[(2R, 5R) -5-methyl-2- [[(3R) -3-methylmorpholine-4-yl] methyl] piperazine-1-yl] etanone dihydrochloride

HCl 4M in dioxane (2 ml, 8 mmol, equivalent: 100), (2R, 5S) -tert-butyl 4- (2- (6- (4-fluorobenzyl) -3- ( Hydroxymethyl) -3-methylindolin-1-yl) -2-oxoethyl) -2-methyl-5-(((R) -3-methylmorpholino) methyl) piperazine-1-carboxylate (50 mg, 80 μmol, equivalent) 1) Was added to the clear solution, and the reaction mixture was stirred at room temperature for 3 hours. The precipitate was filtered off, washed thoroughly with diethyl ether (very hygroscopic), dried in vacuum and 1- (6- (4-fluorobenzyl) -3- (hydroxymethyl) -3. -Methylindrin-1-yl) -2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazine-1-yl) etanone dihydrochloride (43 mg, 89.9%) was obtained as an orange solid. MS: m / z (M + H) ⁺ = 525.3

Monomer D
4-(6- (4-Fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) Acetyl) Indolin-3-Carboxamide) Dihydrochloride 4-(1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R)) -3-Methylmorpholino) methyl) piperazin-1-yl) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxamide) benzoic acid (28 mg, 36.9 μmol, equivalent: 1), Stirred overnight at room temperature with HCl in 4M (0.75ml, 3 mmol, equivalent: 81.2) of dioxane in ethyl acetate (0.75ml). The precipitated solid is filtered off, washed with diethyl ether and dried under high vacuum to allow 4- (6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)-)-. 5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) dihydrochloride benzoate (20 mg, 74.1%) as a white solid Obtained. MS: m / z (M + H) ⁺ = 658.3

Monomer E
2-(6- (4-Fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1) -Il) Acetyl) Indolin-3-Carboxamide) Dihydrochloride 2- (1-(2-((2S, 5R) -4- (tert-butoxycarbonyl) -5-methyl-2-(((R)-)- 3-Methylmorpholino) methyl) piperazin-1-yl) acetyl) -6- (4-fluorobenzyl) -3-methylindrin-3-carboxamide) acetic acid (50 mg, 71.9 μmol, equivalent: 1), ethyl ether In (1 ml), it was stirred with HCl in 4 M (1 ml, 4 mmol, equivalent: 55.7) of dioxane overnight at room temperature. The precipitated solid is filtered off, washed with diethyl ether and dried under high vacuum to give 2- (6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R)-)-. 5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) dihydrochloride (39 mg, 81.2%) obtained as a white solid rice field. MS: m / z (M + H) ⁺ = 596.3

ａ）（Ｓ）−１−ベンジル ２−ｔｅｒｔ−ブチル イソインドリン−１，２−ジカルボキシレート

２５ｍＬの丸底フラスコで、（１Ｓ）−２−ｔｅｒｔ−ブトキシカルボニルイソインドリン−１−カルボン酸（ＣＡＳ １０９３６５１−９３−４；２２０ｍｇ、８３６μｍｏｌ、当量：１）及び重炭酸カリウム（１６７ｍｇ、１．６７ｍｍｏｌ、当量：２）をＤＭＦ（３ｍｌ）と合わせた。臭化ベンジル（２１４ｍｇ、１４９μｌ、１．２５ｍｍｏｌ、当量：１．５）を添加した。反応混合物を、室温で２時間撹拌し、明褐色の懸濁液を形成した。反応混合物を酢酸エチルに注ぎ、希釈水性塩酸及びブラインで抽出した。有機層を、Ｎａ_２ＳＯ_４で乾燥させ、真空中で濃縮した。粗物質をフラッシュクロマトグラフィー（シリカゲル、１２ｇ、０％から４０％ヘプタン中ＥｔＯＡｃ）により精製した：１９４ｍｇの無色の油、ＭＳ：ｍ／ｚ（Ｍ−Ｃ_６Ｈ_５ＣＨ_２ＯＣＯ）^＋＝２１８．１。 Intermediate 1
(S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl ) Isoindoline-1-carboxylic acid

a) (S) -1-benzyl 2-tert-butyl isoindoline-1,2-dicarboxylate

In a 25 mL round bottom flask, (1S) -2-tert-butoxycarbonyl isoindoline-1-carboxylic acid (CAS 1093651-93-4; 220 mg, 836 μmol, equivalent: 1) and potassium carbonate (167 mg, 1.67 mmol). Equivalent: 2) was combined with DMF (3 ml). Benzyl bromide (214 mg, 149 μl, 1.25 mmol, equivalent: 1.5) was added. The reaction mixture was stirred at room temperature for 2 hours to form a light brown suspension. The reaction mixture was poured into ethyl acetate and extracted with diluted aqueous hydrochloric acid and brine. The organic layer was _{dried over Na 2} SO ₄ and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 12g, 0 40% in heptane from% EtOAc): colorless oil of _{194mg, MS: m / z (} M-C 6 H 5 CH 2 OCO) + = 218. 1.

５０ｍＬの丸底フラスコで、（Ｓ）−１−ベンジル ２−ｔｅｒｔ−ブチル イソインドリン−１，２−ジカルボキシレート（１９０ｍｇ、５３８μｍｏｌ、当量：１）をジクロロメタン（６ｍｌ）と合わせた。トリフルオロ酢酸（３ｍｌ）を添加した。混合物を室温で２時間撹拌し、その際、明黄色の溶液が形成された。反応混合物を真空中で濃縮し、２３５ｍｇの黄色の油を得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝２５４．２ b) (S) -Benzyl isoindoline-1-carboxylate 2,2,2-trifluoroacetic acid

In a 50 mL round bottom flask, (S) -1-benzyl 2-tert-butyl isoindoline-1,2-dicarboxylate (190 mg, 538 μmol, equivalent: 1) was combined with dichloromethane (6 ml). Trifluoroacetic acid (3 ml) was added. The mixture was stirred at room temperature for 2 hours, when a bright yellow solution was formed. The reaction mixture was concentrated in vacuo to give 235 mg of yellow oil. MS: m / z (M + H) ⁺ = 254.2

２５ｍＬの丸底フラスコで、（Ｓ）−ベンジル イソインドリン−１−カルボキシレート ２，２，２−トリフルオロアセテート（２３０ｍｇ、５２６μｍｏｌ、当量：１），（Ｓ）−２−（ｔｅｒｔ−ブトキシカルボニルアミノ）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）酢酸（１５０ｍｇ、５７９μｍｏｌ、当量：１．１）及びＤＩＰＥＡ（３４０ｍｇ、４５９μｌ、２．６３ｍｍｏｌ、当量：５）をＤＭＦ（５ｍｌ）と合わせ、その後、ＨＡＴＵ（２６０ｍｇ、６８４μｍｏｌ、当量：１．３）を添加した。混合物を室温で３時間撹拌した。反応混合物を酢酸エチルに注ぎ、希釈ＨＣｌ及びブラインで抽出した。有機層をＮａ２ＳＯ４上で乾燥させ、真空中で濃縮した。粗物質をフラッシュクロマトグラフィー（シリカゲル、１２ｇ、０％から５０％ヘプタン中酢酸エチル）により精製して、１８８ｍｇの白色の固体を得た。ＭＳ：ｍ／ｚ（Ｍ−Ｃ_４Ｈ_８）^＋＝４３９．３ c) (S) -benzyl 2-((S) -2-((tert-butoxycarbonyl) amino) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-1-carboxylate

In a 25 mL round bottom flask, (S) -benzyl isoindoline-1-carboxylate 2,2,2-trifluoroacetate (230 mg, 526 μmol, equivalent: 1), (S) -2- (tert-butoxycarbonylamino) ) -2- (Tetrahydro-2H-pyran-4-yl) acetic acid (150 mg, 579 μmol, equivalent: 1.1) and DIPEA (340 mg, 459 μl, 2.63 mmol, equivalent: 5) were combined with DMF (5 ml). Then HATU (260 mg, 684 μmol, equivalent: 1.3) was added. The mixture was stirred at room temperature for 3 hours. The reaction mixture was poured into ethyl acetate and extracted with diluted HCl and brine. The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 12 g, 0% to 50% ethyl acetate in heptane) to give 188 mg of a white solid. MS: m / z ( _{MC 4} H ₈ ) ⁺ = 439.3

５０ｍＬの丸底フラスコで、（Ｓ）−ベンジル ２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）アミノ）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキシレート（１８５ｍｇ、３７４μｍｏｌ、当量：１）をジクロロメタン（６ｍｌ）と合わせて、無色の溶液を得た。トリフルオロ酢酸（３ｍｌ）を添加した。反応混合物を室温で１時間撹拌し、その後真空中で濃縮した：２８３ｍｇの無色の油。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝３９５．３ d) (S) -Benzyl 2-((S) -2-amino-2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-1-carboxylate 2,2,2-trifluoroacetate

In a 50 mL round bottom flask, (S) -benzyl 2-((S) -2-((tert-butoxycarbonyl) amino) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-1 -Carboxylate (185 mg, 374 μmol, equivalent: 1) was combined with dichloromethane (6 ml) to give a colorless solution. Trifluoroacetic acid (3 ml) was added. The reaction mixture was stirred at room temperature for 1 hour and then concentrated in vacuo: 283 mg of colorless oil. MS: m / z (M + H) ⁺ = 395.3

２５ｍＬの丸底フラスコで、（Ｓ）−ベンジル ２−（（Ｓ）−２−アミノ−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキシレート ２，２，２−トリフルオロアセテート（２８０ｍｇ、３６９μｍｏｌ、当量：１．００）及びＢｏｃ−Ｎ−メチル−Ｌ−ａｌａｎｉｎｅ（８２．５ｍｇ、４０６μｍｏｌ、当量：１．１）をＤＭＦ（４ｍｌ）と合わせて、黄色の溶液を得た。ＤＩＰＥＡ（２８６ｍｇ、３８７μｌ、２．２１ｍｍｏｌ、当量：６）及びＨＡＴＵ（１６８ｍｇ、４４３μｍｏｌ、当量：１．２）を添加した。反応混合物を室温で一晩撹拌した。反応混合物を酢酸エチルに注ぎ、希釈ＨＣｌ、ブラインで抽出した。有機層をＮａ２ＳＯ４上で乾燥させ、真空中で濃縮した。粗物質をフラッシュクロマトグラフィー（シリカゲル、１２ｇ、０％から１００％ヘプタン中酢酸エチル）により精製した：１６６ｍｇの白色の固体。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝５８０．４ e) (S) -Benzyl 2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-) Il) Acetyl) Isoindoline-1-carboxylate

In a 25 mL round bottom flask, (S) -benzyl 2-((S) -2-amino-2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindoline-1-carboxylate 2,2,2 -Trifluoroacetate (280 mg, 369 μmol, equivalent: 1.00) and Boc-N-methyl-L-alane (82.5 mg, 406 μmol, equivalent: 1.1) combined with DMF (4 ml), a yellow solution Got DIPEA (286 mg, 387 μl, 2.21 mmol, equivalent: 6) and HATU (168 mg, 443 μmol, equivalent: 1.2) were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into ethyl acetate and extracted with diluted HCl and brine. The organic layer was dried over Na2SO4 and concentrated in vacuo. A 166 mg white solid obtained by purifying the crude material by flash chromatography (silica gel, 12 g, 0% to 100% ethyl acetate in heptane). MS: m / z (M + H) ⁺ = 580.4

５０ｍＬの丸底フラスコで、（Ｓ）−ベンジル ２−（（Ｓ）−２−（（Ｓ）−２−（（ｔｅｒｔ−ブトキシカルボニル）（メチル）アミノ）プロパンアミド）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）イソインドリン−１−カルボキシレート（１６０ｍｇ、２７６μｍｏｌ、当量：１）をメタノール（１０ｍｌ）中に溶解した。アルゴンを反応混合物中に５分間吹き込んだ。パラジウム炭（Ｐｄ−Ｃ １０％、２９．４ｍｇ、２７．６μｍｏｌ、当量：０．１）を添加した。水素を５分間吹き込んだ。反応混合物を水素雰囲気下で２時間撹拌した。反応混合物をセライトを通して濾過し、真空中で濃縮し、１６４ｍｇの黄色の油を得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝４９０．２５５ f) (S) -2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) ) Acetyl) Isoindoline-1-carboxylic acid

In a 50 mL round bottom flask, (S) -benzyl 2-((S) -2-((S) -2-((tert-butoxycarbonyl) (methyl) amino) propanamide) -2- (tetrahydro-2H) -Pyran-4-yl) acetyl) isoindoline-1-carboxylate (160 mg, 276 μmol, equivalent: 1) was dissolved in methanol (10 ml). Argon was blown into the reaction mixture for 5 minutes. Palladium on carbon (Pd-C 10%, 29.4 mg, 27.6 μmol, equivalent: 0.1) was added. Hydrogen was blown in for 5 minutes. The reaction mixture was stirred under a hydrogen atmosphere for 2 hours. The reaction mixture was filtered through Celite and concentrated in vacuo to give 164 mg of yellow oil. MS: m / z (M + H) ⁺ = 490.255

ａ）（Ｓ）−６−ニトロイソインドリン−１−カルボン酸

（Ｓ）−イソインドリン−１−カルボン酸 ヒドロクロリド（２ｇ、１０ｍｍｏｌ、当量：１）を濃縮したＨ_２ＳＯ_４（９．８３ｇ、５．３４ｍｌ、１００ｍｍｏｌ、当量：１０）に添加し、混合物を撹拌して褐色の溶液を得た。反応混合物を−１５℃に冷却し（氷／ＮａＣｌ浴）、その後、６５％の水性硝酸（１．９４ｇ、１．３９ｍｌ、２０ｍｍｏｌ、当量：２）を添加した。反応混合物を２時間撹拌し、同時に−１５℃から室温へゆっくりと温めて、明黄色の溶液を得た。ＬＣ−ＭＳは、反応が完結したことを示した。反応混合物を氷上に注ぎ、１７ｍｌの２５％アンモニア水を０℃で滴下して、褐色の溶液（ｐＨ８）を得た。反応混合物を真空中で濃縮し、１５．５ｇのダークグレーの固体を得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝２０９．１ Intermediate 2
tert-butyl ((S) -1-(((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindoline-2-yl) -2-) Oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate

a) (S) -6-nitroisoindoline-1-carboxylic acid

(S) -Isoindoline-1-carboxylic acid Hydrochloride (2 g, 10 mmol, equivalent: 1) is _{added to concentrated H 2} SO ₄ (9.83 g, 5.34 ml, 100 mmol, equivalent: 10) and the mixture is added. Stirring gave a brown solution. The reaction mixture was cooled to −15 ° C. (ice / NaCl bath) and then 65% aqueous nitric acid (1.94 g, 1.39 ml, 20 mmol, equivalent: 2) was added. The reaction mixture was stirred for 2 hours and at the same time slowly warmed from −15 ° C. to room temperature to give a bright yellow solution. LC-MS showed that the reaction was complete. The reaction mixture was poured onto ice and 17 ml of 25% aqueous ammonia was added dropwise at 0 ° C. to give a brown solution (pH 8). The reaction mixture was concentrated in vacuo to give 15.5 g of a dark gray solid. MS: m / z (M + H) ⁺ = 209.1

ＴＨＦ（８０ｍｌ）及び水（８０ｍｌ）中（Ｓ）−６−ニトロイソインドリン−１−カルボン酸（１５．５ｇ、９．９ｍｍｏｌ、当量：１）及び重炭酸ナトリウム（３．３３ｇ、３９．６ｍｍｏｌ、当量：４）の溶液に、Ｂｏｃ無水物（４．３２ｇ、４．６ｍｌ、１９．８ｍｍｏｌ、当量：２）を添加した。反応混合液を室温で一晩撹拌した。その後、飽和Ｎａ_２ＣＯ_３水溶液を添加し、混合物をＥｔＯＡｃで２回抽出した。２Ｎ ａｑ． ＨＣｌを水層（ｐＨ１）に滴下し、水層をＥｔＯＡｃで抽出した。有機層を、Ｎａ_２ＳＯ_４で乾燥させ、真空中で濃縮し、２．１５ｇの褐色の泡を得て、それ以上精製することなく次の工程に使用した。 b) (S) -2- (tert-butoxycarbonyl) -6-nitroisoindoline-1-carboxylic acid

In THF (80 ml) and water (80 ml) (S) -6-nitroisoindoline-1-carboxylic acid (15.5 g, 9.9 mmol, equivalent: 1) and sodium bicarbonate (3.33 g, 39.6 mmol, Boc anhydride (4.32 g, 4.6 ml, 19.8 mmol, equivalent: 2) was added to the solution of equivalent: 4). The reaction mixture was stirred at room temperature overnight. Then saturated _{aqueous Na 2} CO ₃ solution was added and the mixture was extracted twice with EtOAc. 2N aq. HCl was added dropwise to the aqueous layer (pH 1) and the aqueous layer was extracted with EtOAc. The organic layer was _{dried over Na 2} SO ₄ and concentrated in vacuo to give 2.15 g of brown foam for use in the next step without further purification.

ＥｔＯＡｃ（１０ｍｌ）中（Ｓ）−２−（ｔｅｒｔ−ブトキシカルボニル）−６−ニトロイソインドリン−１−カルボン酸（２．１５ｇ、６．９７ｍｍｏｌ、当量：１）の撹拌された溶液に、２，６−ジフルオロアニリン（１．０８ｇ、８４６μｌ、８．３７ｍｍｏｌ、当量：１．２）、ピリジン（５ｍｌ）及びＥｔＯＡｃ中Ｔ３Ｐ（８．８８ｇ、８．３ｍｌ、１３．９ｍｍｏｌ、当量：２）の ５０％溶液を連続して添加した。反応混合物を室温で１時間撹拌して、黄色の溶液を得た。ＬＣ−ＭＳは、反応が完結したことを示した。反応混合物をＥｔＯＡｃに注ぎ、１Ｎ ａｑ． ＨＣｌ及び飽和ブラインで連続して抽出した。有機層を分離し、Ｎａ_２ＳＯ_４で乾燥させ、真空中で濃縮した。粗物質をフラッシュクロマトグラフィー（シリカゲル、８０ｇ、０％から１００％ヘプタン中ＥｔＯＡｃ）により精製して、２．１６ｇの明褐色の固体を得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ−Ｃ_４Ｈ_８）^＋＝３６４．１ c) (S) -tert-butyl 1-((2,6-difluorophenyl) carbamoyl) -6-nitroisoindoline-2-carboxylate

In a stirred solution of (S) -2- (tert-butoxycarbonyl) -6-nitroisoindoline-1-carboxylic acid (2.15 g, 6.97 mmol, equivalent: 1) in EtOAc (10 ml), 2, 50% of 6-difluoroaniline (1.08 g, 846 μl, 8.37 mmol, equivalent: 1.2), pyridine (5 ml) and T3P (8.88 g, 8.3 ml, 13.9 mmol, equivalent: 2) in EtOAc. The solution was added continuously. The reaction mixture was stirred at room temperature for 1 hour to give a yellow solution. LC-MS showed that the reaction was complete. The reaction mixture is poured into EtOAc and 1 N aq. Extracted sequentially with HCl and saturated brine. The organic layer was separated _{, dried over Na 2} SO ₄ and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 80 g, EtOAc in 0% to 100% heptane) to give 2.16 g of a light brown solid. MS: m / z (M + HC ₄ H ₈ ) ⁺ = 364.1

ＣＨ_２Ｃｌ_２（２０ｍｌ）中（Ｓ）−ｔｅｒｔ−ブチル １−（（２，６−ジフルオロフェニル）カルバモイル）−６−ニトロイソインドリン−２−カルボキシレート（２．１ｇ、５．０１ｍｍｏｌ、当量：１）の溶液に、ＴＦＡ（１４．８ｇ、１０ｍｌ、１３０ｍｍｏｌ、当量：２６）を添加した。反応混合物を室温で２時間撹拌した。その後、反応混合物を真空中で濃縮し、残留物をＥｔＯＡｃに溶解し、飽和水性ＮａＨＣＯ_３及び飽和ブラインで連続して抽出した。有機層をＮａ_２ＳＯ_４で乾燥させ、真空中で濃縮し、１．４１ｇの褐色の固体を得て、それ以上精製することなく次の工程に使用した。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝３２０．１ d) (S) -N- (2,6-difluorophenyl) -6-nitroisoindoline-1-carboxamide

CH ₂ Cl ₂ (20 ml) in (S) -tert-butyl 1-((2,6-difluorophenyl) carbamoyl) -6-nitroisoindoline-2-carboxylate (2.1 g, 5.01 mmol, equivalent: TFA (14.8 g, 10 ml, 130 mmol, equivalent: 26) was added to the solution of 1). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then concentrated in vacuo and the residue was dissolved in EtOAc and _{extracted sequentially with saturated aqueous NaHCO 3} and saturated brine. The organic layer was _{dried over Na 2} SO ₄ and concentrated in vacuo to give 1.41 g of a brown solid which was used in the next step without further purification. MS: m / z (M + H) ⁺ = 320.1

ＤＭＦ（１０ｍｌ）中（Ｓ）−Ｎ−（２，６−ジフルオロフェニル）−６−ニトロイソインドリン−１−カルボキサミド（１．４ｇ、４．３９ｍｍｏｌ、当量：１），（Ｓ）−２−（ｔｅｒｔ−ブトキシカルボニルアミノ）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）酢酸（１．１９ｇ、４．６ｍｍｏｌ、当量：１．０５）及びＤＩＰＥＡ（１．１３ｇ、１．５３ｍｌ、８．７７ｍｍｏｌ、当量：２）の溶液に、ＨＡＴＵ（２．１７ｇ、５．７ｍｍｏｌ、当量：１．３）を添加した。反応混合物を室温で一晩撹拌した。その後、反応混合物をＥｔＯＡｃに注ぎ、０．５Ｎ ａｑ． ＨＣｌ及び飽和ブラインで連続して抽出した。有機層を分離し、Ｎａ_２ＳＯ_４で乾燥させ、真空中で濃縮した。粗物質をフラッシュクロマトグラフィー（シリカゲル、８０ｇ、ヘプタン／ＣＨ_２Ｃｌ_２／ＭｅＯＨ）により精製して、２．２７ｇの黄色の泡を得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝５６１．２ e) tert-butyl ((S) -2-((S) -1-((2,6-difluorophenyl) carbamoyl) -6-nitroisoindoline-2-yl) -2-oxo-1- (tetrahydro) -2H-pyran-4-yl) ethyl) carbamate

In DMF (10 ml) (S) -N- (2,6-difluorophenyl) -6-nitroisoindoline-1-carboxamide (1.4 g, 4.39 mmol, equivalent: 1), (S) -2- ( tert-butoxycarbonylamino) -2- (tetrahydro-2H-pyran-4-yl) acetic acid (1.19 g, 4.6 mmol, equivalent: 1.05) and DIPEA (1.13 g, 1.53 ml, 8.77 mmol) , Equivalent: 2), HATU (2.17 g, 5.7 mmol, equivalent: 1.3) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture is then poured into EtOAc and 0.5 N aq. Extracted sequentially with HCl and saturated brine. The organic layer was separated _{, dried over Na 2} SO ₄ and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 80 g, heptane / CH ₂ Cl ₂ / MeOH) to give 2.27 g of yellow foam. MS: m / z (M + H) ⁺ = 561.2

ＣＨ_２Ｃｌ_２（１２ｍｌ）中ｔｅｒｔ−ブチル（（Ｓ）−２−（（Ｓ）−１−（（２，６−ジフルオロフェニル）カルバモイル）−６−ニトロイソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）カルバメート（２．２７ｇ、４．０５ｍｍｏｌ、当量：１）の溶液にＴＦＡ（９．２４ｇ、６．２４ｍｌ、８１ｍｍｏｌ、当量：２０）を添加し、反応混合物を室温で２時間撹拌した。反応混合物を真空中で濃縮し、残留物をＥｔＯＡｃに溶解し、水性ＮａＨＣＯ_３及び飽和ブラインで連続して抽出した。有機層を分離し、Ｎａ_２ＳＯ_４で乾燥させ、真空中で濃縮して、１．６８ｇの暗黄色の固体を得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝４６１．２ f) (S) -2-((S) -2-amino-2- (tetrahydro-2H-pyran-4-yl) acetyl) -N- (2,6-difluorophenyl) -6-nitroisoindoline- 1-Carboxamide

Tert-Butyl ((S) -2-((S) -1-((2,6-difluorophenyl) carbamoyl) -6-nitroisoindoline-2-yl) -2-yl in CH ₂ Cl _{2 (12 ml))} TFA (9.24 g, 6.24 ml, 81 mmol, equivalent: 20) in a solution of oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) carbamate (2.27 g, 4.05 mmol, equivalent: 1). Was added and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo and the residue was dissolved in EtOAc and _{extracted sequentially with aqueous NaHCO 3} and saturated brine. The organic layer was separated _{, dried over Na 2} SO ₄ and concentrated in vacuo to give 1.68 g of a dark yellow solid. MS: m / z (M + H) ⁺ = 461.2

ＤＭＦ（８ｍｌ）中（Ｓ）−２−（（Ｓ）−２−アミノ−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）アセチル）−Ｎ−（２，６−ジフルオロフェニル）−６−ニトロイソインドリン−１−カルボキサミド（１．６８ｇ、３．６５ｍｍｏｌ、当量：１）、ＢＯＣ−ＭＥＡＬＡ−ＯＨ（８１６ｍｇ、４．０１ｍｍｏｌ、当量：１．１）及びＤＩＰＥＡ（１．４１ｇ、１．９１ｍｌ、１０．９ｍｍｏｌ、当量：３）の溶液に、ＨＡＴＵ（１．８ｇ、４．７４ｍｍｏｌ、当量：１．３）を添加した。反応混合物を室温で４時間撹拌した。ＬＣ−ＭＳ、ＴＬＣは、反応が完結したことを示した。反応混合物をＥｔＯＡｃに注ぎ、０．５Ｎ ａｑ． ＨＣｌ及び飽和ブラインで連続して抽出した。有機層を分離し、Ｎａ_２ＳＯ_４で乾燥させ、真空中で濃縮した。粗物質をフラッシュクロマトグラフィー（シリカゲル、８０ｇ、ヘプタン／ＣＨ_２Ｃｌ_２／ＭｅＯＨ）により精製して、２．０ｇのオフホワイトの泡を得た。ＭＳ：ｍ／ｚ（Ｍ−Ｈ）⁻＝６４４．４ g) tert-butyl ((S) -1-(((S) -2-((S) -1-((2,6-difluorophenyl) carbamoyl) -6-nitroisoindoline-2-yl)-)- 2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate

In DMF (8 ml) (S) -2-((S) -2-amino-2- (tetrahydro-2H-pyran-4-yl) acetyl) -N- (2,6-difluorophenyl) -6-nitro Isoindoline-1-carboxamide (1.68 g, 3.65 mmol, equivalent: 1), BOC-MEALA-OH (816 mg, 4.01 mmol, equivalent: 1.1) and DIPEA (1.41 g, 1.91 ml, 10) HATU (1.8 g, 4.74 mmol, equivalent: 1.3) was added to a solution of .9 mmol, equivalent: 3). The reaction mixture was stirred at room temperature for 4 hours. LC-MS and TLC showed that the reaction was complete. The reaction mixture was poured into EtOAc and 0.5 N aq. Extracted sequentially with HCl and saturated brine. The organic layer was separated _{, dried over Na 2} SO ₄ and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 80 g, heptane / CH ₂ Cl ₂ / MeOH) to give 2.0 g of off-white foam. MS: m / z (MH) ^- = 644.4

メタノール（５ｍｌ）中ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−１−（（２，６−ジフルオロフェニル）カルバモイル）−６−ニトロイソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート（２ｇ、３．１ｍｍｏｌ、当量：１）の溶液を、アルゴンで１０分間パージした。１０％Ｐｄ−Ｃ（３３０ｍｇ、３１０μｍｏｌ、当量：０．１）を添加した。反応混合物をＨ_２で１０分間パージし、その後、Ｈ_２のバルーン下で１時間撹拌した。ＴＬＣは反応が完結したことを示した。反応混合物をセライトを通して濾過した。９ｇのシリカゲルを濾液に添加し、混合物を真空中で濃縮した。粗物質をフラッシュクロマトグラフィー（シリカゲル、８０ｇ、０％から１０％ＣＨ_２Ｃｌ_２中ＭｅＯＨ）により精製して、１．５７ｇの白色の固体を得た。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６１６． h) tert-butyl ((S) -1-(((S) -2-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindoline-2-yl)-)- 2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate

Tert-Butyl ((S) -1-(((S) -2-((S) -1-((2,6-difluorophenyl) carbamoyl) -6-nitroisoindoline-2-) in methanol (5 ml)) Il) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate (2 g, 3.1 mmol, equivalent: 1) The solution was purged with argon for 10 minutes. 10% Pd-C (330 mg, 310 μmol, equivalent: 0.1) was added. The reaction mixture was _{purged with H 2} for 10 minutes and then stirred under a balloon of _{H 2 for 1 hour.} TLC showed that the reaction was complete. The reaction mixture was filtered through Celite. 9 g of silica gel was added to the filtrate and the mixture was concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 80 g, 0% to 10% _{MeOH in CH 2} Cl ₂ ) to give 1.57 g of a white solid. MS: m / z (M + H) ⁺ = 616.

（Ｓ）−イソインドリン−１−カルボン酸 ヒドロクロリドの代わりに（Ｓ）−１，２，３，４−テトラヒドロイソキノリン−３−カルボン酸から出発して、中間体２と同様に標題化合物を調製した。黄色の固体。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝６３０．３ Intermediate 3
tert-butyl ((S) -1-(((S) -2-((S) -7-amino-3-((2,6-difluorophenyl) carbamoyl) -3,4-dihydroisoquinoline-2 ( 1H) -yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate

(S) -Isoindoline-1-carboxylic Acid Starting with (S) -1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid instead of Hydrochloride, prepare the title compound as in Intermediate 2. bottom. Yellow solid. MS: m / z (M + H) ⁺ = 630.3

ａ）ｔｅｒｔ−ブチル（（２Ｓ）−１−（（（１Ｓ）−２−（５−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート

（Ｓ）−６−ニトロイソインドリン−１−カルボン酸の代わりに５−ニトロイソインドリン−１−カルボン酸 ヒドロクロリドから出発して、中間体２工程（ｂ）−（ｈ）と同様に、標題化合物を調製した。褐色の固体。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）⁻＝６１４．５ Intermediate 4
tert-butyl ((S) -1-(((S) -2-((S) -5-amino-1-((2,6-difluorophenyl) carbamoyl) isoindoline-2-yl) -2-) Oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate

a) tert-butyl ((2S) -1-(((1S) -2-(5-amino-1-((2,6-difluorophenyl) carbamoyl) isoindoline-2-yl) -2-oxo-) 1- (Tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate

(S) Starting from 5-nitroisoindoline-1-carboxylic acid hydrochloride instead of -6-nitroisoindoline-1-carboxylic acid, intermediate 2 steps (b)-(h) as well as the title. Compounds were prepared. Brown solid. MS: m / z (M + H) ^- = 614.5

ｔｅｒｔ−ブチル（（２Ｓ）−１−（（（１Ｓ）−２−（５−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメートを、キラルＨＰＬＣ（カラム：Ｒｅｐｒｏｓｉｌ Ｃｈｉｒａｌ−ＮＲ；溶出剤：ＮＨ_４ＯＡｃ／ＥｔＯＨ／ヘプタンのグラジエント）により分離し、以下を得た：
（＋）−エピマー、保持時間＝１１分、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｒ）−５−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート。黄色の固体。ＭＳ：ｍ／ｚ（Ｍ−Ｈ）⁻＝６１４．５。
（−）−エピマー、保持時間＝２１分、ｔｅｒｔ−ブチル（（Ｓ）−１−（（（Ｓ）−２−（（Ｓ）−５−アミノ−１−（（２，６−ジフルオロフェニル）カルバモイル）イソインドリン−２−イル）−２−オキソ−１−（テトラヒドロ−２Ｈ−ピラン−４−イル）エチル）アミノ）−１−オキソプロパン−２−イル）（メチル）カルバメート。オフホワイトの固体。ＭＳ：ｍ／ｚ（Ｍ−Ｈ）⁻＝６１４．５。 b) tert-butyl ((S) -1-(((S) -2-((S) -5-amino-1-((2,6-difluorophenyl) carbamoyl) isoindoline-2-yl)-)- 2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate

tert-butyl ((2S) -1-(((1S) -2-(5-amino-1-((2,6-difluorophenyl) carbamoyl) isoindoline-2-yl) -2-oxo-1-yl) (Tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate, chiral HPLC (column: Reprosil Cyral-NR; eluent: NH ₄ OAc / EtOH / Separation by heptane gradient) and obtained:
(+)-Epimer, retention time = 11 minutes, tert-butyl ((S) -1-(((S) -2-((R) -5-amino-1-((2,6-difluorophenyl)) Carbamoyl) Isoindoline-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate. Yellow solid. MS: m / z (MH) ⁻ = 614.5.
(-)-Epimer, retention time = 21 minutes, tert-butyl ((S) -1-(((S) -2-((S) -5-amino-1-((2,6-difluorophenyl)) Carbamoyl) Isoindoline-2-yl) -2-oxo-1- (tetrahydro-2H-pyran-4-yl) ethyl) amino) -1-oxopropan-2-yl) (methyl) carbamate. Off-white solid. MS: m / z (MH) ⁻ = 614.5.

（Ｓ）−２−（ｔｅｒｔ−ブトキシカルボニルアミノ）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）酢酸の代わりにＮ−Ｂｏｃ−Ｏ−メチル−Ｌ−スレオニンを使用して、中間体２と同様に標題化合物を調製した。白色の固体。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝５９０．２７ Intermediate 5
tert-Butyl ((S) -1-(((2S, 3R) -1-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindoline-2-yl)- 3-Methoxy-1-oxobutan-2-yl) amino) -1-oxopropan-2-yl) (methyl) carbamate

Intermediate 2 using N-Boc-O-methyl-L-threonine instead of (S) -2- (tert-butoxycarbonylamino) -2- (tetrahydro-2H-pyran-4-yl) acetic acid The title compound was prepared in the same manner as in the above. White solid. MS: m / z (M + H) ⁺ = 590.27

（Ｓ）−２−（ｔｅｒｔ−ブトキシカルボニルアミノ）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）酢酸の代わりに Ｂｏｃ−Ｌ−イソロイシンを使用して、中間体２と同様に標題化合物を調製した。明黄色の固体。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝５８８．３ Intermediate 6
tert-Butyl ((S) -1-(((2S, 3S) -1-((S) -6-amino-1-((2,6-difluorophenyl) carbamoyl) isoindoline-2-yl)- 3-Methyl-1-oxopentan-2-yl) amino) -1-oxopropan-2-yl) (methyl) carbamate

(S) Boc-L-isoleucine instead of -2- (tert-butoxycarbonylamino) -2- (tetrahydro-2H-pyran-4-yl) acetic acid was used to obtain the title compound as in Intermediate 2. Prepared. A bright yellow solid. MS: m / z (M + H) ⁺ = 588.3

（Ｓ）−２−（ｔｅｒｔ−ブトキシカルボニルアミノ）−２−（テトラヒドロ−２Ｈ−ピラン−４−イル）酢酸の代わりに Ｂｏｃ−Ｌ−ｔｅｒｔ−ロイシンを使用して、中間体２と同様に標題化合物を調製した。明黄色の油。ＭＳ：ｍ／ｚ（Ｍ＋Ｈ）^＋＝５８８．３ Intermediate 7
tert-Butyl N-[(1S) -2-[[(1S) -1-[(1S) -6-amino-1-[(2,6-difluorophenyl) carbamoyl] isoindoline-2-carbonyl]- 2,2-Dimethyl-propyl] amino] -1-methyl-2-oxo-ethyl] -N-methyl-carbamate

(S) Using Boc-L-tert-leucine instead of -2- (tert-butoxycarbonylamino) -2- (tetrahydro-2H-pyran-4-yl) acetic acid, the title is similar to Intermediate 2. Compounds were prepared. Light yellow oil. MS: m / z (M + H) ⁺ = 588.3

Claims (15)

Formula I

[During the ceremony,
A is

Selected from the group consisting of
n is 0 or 1 and
R ₁ is selected from the group consisting of H, halogen or C _1-6-alkyl.
R ₂ is selected from the group consisting of H, halogen or C _1-6-alkyl.
R ₃ is selected from the group consisting of H, halogen or C _1-6-alkyl.
R _{4 is, C 1-6} - _{alkyl, C 1-6} - alkoxy _{-C 1-6} - alkyl or _{C 3-6} - is selected from the group consisting of heterocycloalkyl,
R ₅ is selected from the group consisting of H, halogen or C _1-6-alkyl.
R ₆ is selected from the group consisting of H, halogen or C _1-6-alkyl.
L is

Selected from the group consisting of
m is 1, 2 or 3
k is 1, 2 or 3]
Compound or pharmaceutically acceptable salt thereof. The compound of formula I according to claim 1, wherein R _{1 is F.} A is A-1, R ₂ and R ₃ are F, and R ₄ is

The compound of formula I according to claim 1 or 2, selected from the group consisting of. A is A-2, and either _{R 5} is F, or A is A-4, and _{R 6} is F, the compound of formula I according to claim 1 or 2. The compound of formula I according to any one of claims 1 to 4, wherein L is L-1, L-2 or L-3. The compound of formula I according to any one of claims 1 to 4, wherein L is L-4 or L-6. The compound of formula I according to any one of claims 1 to 4, wherein L is L-5 and k is 3. N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-) 2H-pyran-4-yl) acetyl) isoindolin-5-yl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-methyl-2- ( ((R) -3-Methylmorpholino) Methyl) Piperazin-1-yl) Acetyl) Indolin-3-Carboxamide Trihydrochloride,
(3R) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino)) Propanoyl] amino] -2- (oxan-4-yl) acetyl] -1,3-dihydroisoindole-5-yl] amino] -2-oxoethyl] -6-[(4-fluorophenyl) methyl] -3 -Methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] piperazin-1-yl] acetyl] -2H-indole -3-Carboxamide trihydrochloride,
(3S) -N- [2-[[(3S) -3-[(2,6-difluorophenyl) carbamoyl] -2-[(2S) -2-[[(2S) -2- (methylamino)) Propanoyl] amino] -2- (oxan-4-yl) acetyl] -1,3-dihydroindole-5-yl] amino] -2-oxoethyl] -6-[(4-fluorophenyl) methyl] -3- Methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholin-4-yl] methyl] piperazine-1-yl] acetyl] -2H-indole- 3-Carboxamide trihydrochloride,
(3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2- (methylamino) propanoyl] amino] butanoyl] -7-[[2-[[(3S)- 6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] ] Piperazin-1-yl] acetyl] -2H-indole-3-carbonyl] amino] acetyl] amino] -N-[(1R) -1,2,3,4-tetrahydronaphthalene-1-yl] -3, 4-Dihydro-1H-isoquinolin-3-carboxamide trihydrochloride,
(3S) -2-[(2S) -3,3-dimethyl-2-[[(2S) -2- (methylamino) propanoyl] amino] butanoyl] -7-[[2-[[(3R)- 6-[(4-fluorophenyl) methyl] -3-methyl-1- [2-[(2R, 5R) -5-methyl-2-[[(3R) -3-methylmorpholine-4-yl] methyl] ] Piperazin-1-yl] acetyl] -2H-indole-3-carbonyl] amino] acetyl] amino] -N-[(1R) -1,2,3,4-tetrahydronaphthalene-1-yl] -3, 4-Dihydro-1H-isoquinolin-3-carboxamide trihydrochloride,
(3S) -2-((S) -3,3-dimethyl-2-((S) -2- (methylamino) propanoamide) butanoyl) -7- (6- (4-fluorobenzyl) -3-3 Methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) -N- ((R) -1,2,3,4-tetrahydronaphthalene-1-yl) -1,2,3,4-tetrahydroisoquinolin-3-carboxamide trihydrochloride,
(3S) -2-((S) -3,3-dimethyl-2-((S) -2- (methylamino) propanoamide) butanoyl) -7- (2- (6- (4-fluorobenzyl)) -3-Methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) Acetamide) -N-((R) -1,2,3,4-tetrahydronaphthalene-1-yl) -1,2,3,4-tetrahydroisoquinolin-3-carboxamide trihydrochloride,
(3S) -N- (2,6-difluorophenyl) -7- (2- (6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R) -5-methyl-) 2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide) acetamide) -2-((S) -2-((S) -2- (methyl) Amino) Propanamide) -2- (Tetrahydro-2H-Pyran-4-yl) Acetyl) -1,2,3,4-Tetrahydroisoquinolin-3-carboxamide Trihydrochloride,
(R) -N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl) -2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(R, R, R) -N, N'-(ethane-1,2-diyl) bis (6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5) -Methyl-2-(((R) -3-methylmorpholino) methyl) piperazine-1-yl) acetyl) indoline-3-carboxamide) trihydrochloride,
(S) -N-((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) -6- (4-fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl) -2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide,
(S) -N- (2-(((S) -1-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propane) Amide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2) -((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((2S, 3R) -3-methoxy-2-((S) -2) -(Methylamino) propanamide) butanoyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)-)- 5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((2S, 3S) -3-methyl-2-((S) -2) -(Methylamino) propanamide) pentanoyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)-)- 5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(S) -N- (2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -3,3-dimethyl-2-((S) -2) -(Methylamino) propanamide) butanoyl) isoindrin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)-)- 5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(S) -N- (3-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propane) Amide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-5-yl) amino) -3-oxopropyl) -6- (4-fluorobenzyl) -3-methyl-1- ( 2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
(S) -N- (4-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propane) Amide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindrin-5-yl) amino) -4-oxobutyl) -6- (4-fluorobenzyl) -3-methyl-1- (2) -((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
N-(2-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) amino) -2-oxoethyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R) , 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
N- (2- (4-Fluoro-3-((S) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-) 4-yl) Acetyl) Isoindrin-1-Carboxamide) Benzamide) Ethyl) -6- (4-Fluorobenzyl) -3-Methyl-1-(2-((2R, 5R) -5-Methyl-2- ( ((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide dihydrochloride,
N-(4-(((S) -3-((2,6-difluorophenyl) carbamoyl) -2-((S) -2-((S) -2- (methylamino) propanamide) -2) -(Tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) carbamoyl) phenyl) -6- (4-fluorobenzyl) -3-methyl-1- (2-((2R, 5R)) -5-Methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride,
N- (4- (4-Fluoro-3-((S) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-) 4-Il) Acetyl) Isoindrin-1-Carboxamide) Benzamide) Phenyl) -6- (4-Fluorobenzyl) -3-Methyl-1-(2-((2R, 5R) -5-Methyl-2- ( ((R) -3-methylmorpholino) methyl) piperazin-1-yl) acetyl) indolin-3-carboxamide trihydrochloride, and N1-((S) -3-((2,6-difluorophenyl) carbamoyl)) -2-((S) -2-((S) -2- (methylamino) propanamide) -2- (tetrahydro-2H-pyran-4-yl) acetyl) isoindolin-5-yl) -N3- (3-(6- (4-Fluorobenzyl) -3-methyl-1-(2-((2R, 5R) -5-methyl-2-(((R) -3-methylmorpholino) methyl) piperazin-) The compound according to any one of claims 1 to 7, selected from the group consisting of 1-yl) acetyl) indolin-3-yl) propyl) malonamide trihydrochloride. The compound according to any one of claims 1 to 8, for use as a therapeutically active substance. The compound according to any one of claims 1 to 8, for use in therapeutic and / or prophylactic treatment of cancer. Formula I-a of the dimer compound of formula I

[During the ceremony,
R ₁ is selected from the group consisting of H, halogen or C _1-6-alkyl.
R is

Selected from the group consisting of]
Monomer. As inhibitors of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or function as inhibitors of active caspase proteins that bind to I AP, cancer, in particular improved solid tumors, treatment or control method The compound according to any one of claims 1 to 8 for use as a combination therapy with a bispecific antibody, wherein the bispecific antibody is desceptor 5 (DR5) and fibroblasts. The invention according to any one of claims 1 to 8, which binds to an activating protein (FAP) and comprises at least one DR5-specific antigen-binding site and at least one FAP-specific antigen-binding site. of the compound. In combination with death receptor 5 (DR5) and fibroblast activation protein (FAP) to bind to the bispecific antibody of claim 12, as an inhibitor of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or function as inhibitors of active caspase proteins that bind to I AP, compound of any one of claims 1 to 8. As inhibitors of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or function as inhibitors of active caspase proteins that bind to I AP, combination therapy with bispecific antibody of claim 12 The compound according to any one of claims 1 to 8, wherein the bispecific antibody comprises a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8. To a FAP containing at least one antigen binding site specific for DR5 containing a variable light chain region containing, a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 16. The compound according to any one of claims 1 to 8 , which comprises at least one specific antigen-binding site. In combination with death receptor 5 (DR5) and fibroblast activation protein (FAP) to bind to the bispecific antibody of claim 12, as an inhibitor of SMAC protein that binds to inhibitor of apoptosis protein (IAP), and / or serve as an inhibitor of activated caspase proteins that bind to I AP, a compound of according to any one of claims 1 to 8, bispecific antibody, SEQ ID NO: 7 At least one DR5-specific antigen binding site containing a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 8 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15. The compound according to any one of claims 1 to 8 , which comprises at least one FAP-specific antigen binding site comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

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